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1. sess 55 Secondary Antibody Binding 42 SENSIVINV uns d kke 58 Settled Microspheres ssarrsnsvorovorvsrovoverenrsvereresesveveresesnsne 34 Inl Sense HER 50 Settling Rates sess 53 55 Settling Velocity sese 37 SHIT LIT Occ e reete en 47 Shipping Methods sss 25 29 Silica Adsorption 31 Silica Applications 22 Silica Microspheres 6 22 31 33 39 47 51 54 Simply CellularQ ssesssseennenennnne 11 19 Simply Cellular anti Mouse Compensation Standard 4 Simply Cellular amp Compensation Standard 20 Small Beads sse nens 50 Small COOH Modified Beads sss 32 Small Microspheres seeseseeess 16 44 45 55 SNARE vunne Gene tret ederet ire Ee 13 Sodium Azidg eee 29 Solids Concentration Levels sees 30 Solids Content 55 59 SOMGATION m 40 Spacer AM uei enero nesten 54 SPACErSu eee 20 47 Spectrophotometer sss 36 63 Sta pilit eroi t tene reete dois 9 41 Staining Samples essere 11 Standardization sss 13 23 Sterilizing Microspheres seen 2 SUCKING m 63 Stokes Diameter sse 52 Stokes AW oisi etri ed eek 55 NL RR REEL EN 29 46 47 49 Storage Conditions
2. c eeeeeenveevrvvrvorerrrrorererrsroverenesrsvrvenerenne Continuous Centrifugation 52 COOH Modified Beads 32 33 38 39 46 47 48 49 62 Coulombs per Particle 48 Coupling Antibodies 18 Coupling Efficiency 26 Coupling Process 6 Coupling Strategies 4 5 Covalent Coupling 29 32 Gross Flow Filtration 32 41 44 52 53 55 GroSSlinker avstanden 21 Crosslinking cien udi Ern 7 20 Custom Coupling eene 33 38 Custom Services ereere 18 33 38 GysGliroimelM eeu cente ert cert eet tenes 14 D DET VAN OE tnnt 17 DP 12 Deionized Water sss 43 Denaturatior cm ret eda 36 BLE TEN chet 48 DeSOF OTI cierto rt ir tr re a re tete ers 34 Desorption of Protein sss 46 Diagnostic Test Design 53 DANSET ERE 16 50 Dilute Suspensions sssssssseeerenenennee 59 BIA ETON ER 31 Directed Immobilization seeee 14 Disulfide Bonds sss 53 Divinylbenzene oervrnnrnronvovenrononnvenvnnnnronnvnnenrorvernenner 37 43 DNA EE 10 DNA Adsorption seseses 31 36 39 51 57 DNA Attachment esorerrrrrervrrerervrervervrervvrsvrrversverversvernene 33 35 DNA Binding 2 iine cete c tere eres 39 DNA Hybridization
3. sse 10 23 Antibody Source rt thass 22 Antibody Welglits tite 4 APG Gy senderen 12 B Bactetla i cette eee nete a 58 Bangs Bead Buffers saisonnier 5 Bangs Bead Solution 5 Bead Handling 5 m rmt tm mers 16 Bicarbonate Bute iit ees 29 Binding Capacity 3 29 Binding DNA a et ec tere ets 57 Binding Efficiencies seseennn 3 Binding sslles x rtr tes 45 Binding Oligonucleotides sessssss 57 Binding Polypeptides osoasa 46 Binding Protein esee 32 53 BIOMAg B cete 2 14 17 18 20 21 32 BioMag Immobilization Kit esee 21 BioMag GgPlus ct er dente Rte teet 14 BioMag GPlus Amine sse 18 BioMag amp Plus Carboxyl sene 18 BioMag amp Plus Concanavalin A sss 32 BioMag Plus Wheat Germ Agglutinin 21 sine 19 Boun Binding iut ees 23 Blotin EIUtlori s a m ee 27 Biotinylated Oligo Bound sees 12 Biotinylated Oligonucleotides sss 29 Biotinylated Proteills mes 3 Blocker Goncentration sewsrsvvrververververvrsvenverrrserveerenser 25 40 BlOGKers tee te 30 45 47 52 59 61 C Cali Brati enin n nime ces Calibration Standards Carbodilimide dte tt aaia Carboxylated Beads
4. sss 29 Microsphere Dispersion seen 49 Mobility of Particles soorrrvrrrrrrrrsvrsrsvrsrsvrsveresvenrsnennsnennsenn 62 Molecular Biology 15 Monoclonal Antibodies sse 61 MonodISDersity rre trt re ee er reta 63 Mif EVI o 33 Mounting Mediums seennnnnn 6 N Nephelometers sse 63 Nephelometric Assays sen 57 NHS Iminobiotin sse 37 NAS ESS EN 26 NIST Traceable Particles 9 NitNG Aldiss ann 41 Nitrocellulose Membranes ene 51 Non lonic Surfactant sossonorovororrrrororerrsnsvereresvsrsvonesnsvsvener 32 Nonspecific Binding 22 29 46 59 61 0 OEM Catalog Numbers 40 Oligo Binding sese 26 One Step vs Two Step Coupling sessss 49 Optical Alignment 18 Organic Beads 41 Organic Solvents sss 44 Orientation cette eene 36 39 P Parking Area sesorororrornonrorrosvorrerrerornrsverrorvervenne 26 48 52 57 Particle Concentration sss 59 Particle Counters 63 Particle Density e eer ta 53 Particle Determination 26 34 39 47 58 61 64 Particle Diameter iter 58 Particle olv rr 63 Particle Sizini ciae e te 9 29 Pass Fail Criteria ssssssssseeeenennen 3 PCR Thermocycl
5. sssssseeennnnn 35 DNA IS0latlQI c a a 13 DNA Purification sss 13 36 DNA Sequencing esesseseeeeenennn nennen 37 Drying Beads sss 41 44 Dry Microspheresa 3c rr teet how t eis 31 By EI Em 54 Dyed Microspheres sen 54 Dy Tenminatol D 37 E ED AG ES 27 EDAC Coupling essere 14 62 EIOGUOIWICS saints tore siete ates ent 64 Electrophoretic Mobility Instruments 63 o p ES 37 Equations 26 48 51 52 53 55 56 57 Equilibrium Adsorption sene 33 Ethanolamine resoresvrrrrrrervrreverrervervvesvvssverversverversvernene 27 41 Extreme Temperatures essen 37 F FAP RAO urne ost 10 22 23 F r Qut Red ee eite reet ret ee tees 12 Field Flow Fractionation sene 63 Filter Method 60 ESAO 45 P AO Deserria TS 16 FITC PE Compensation Standard sss 20 Flash Red ihe tem ter hen e ds 3 FloCcilatiori 2 creta 64 Flow FISH cscsesesescsssscscscsesececsesesesavsesesesevececsesesesavseeesasanes 15 Flow Cytometers cecccscsesscscsesecesscsesesesscecscsesecessesesecesaees 17 Flow Cytometric ASSAYS sse 14 Flow Cytometry ssi ceeccescscsscscsscsetscsesseseceesecseseceenees 5 6 20 22 Flow Cytometry Absolute Count Standard 19 Fluores
6. Bangs Laboratories e Ask The Particle Doctor Page 36 your agglutination test anyway Since we know nothing about your antigen we cannot help with ratios of Ag to Ab except to give a general answer It looks as if you have only a fraction of a monolayer maybe 1 4mg IgG m 7mg lgG m 20 of monolayer but still it is possible that you have too much IgG on the surface To get bridging with antigen Ag you must have an Ag with at least two epitopes per molecule If both epitopes on Ag can react with adjacent IgGs on the same microsphere you will get no bridging You must space IgG molecules on the bead surface so Ag reacting with one IgG cannot react with another IgG on the same microsphere You can try using less IgG to coat latex It is recommended that you do a box titration varying both If too much Ag is used then no bridging will occur because each Ag can find only one IgG Prozone or hook effect You could also try higher solids content even 0 5 may be too low for your assay since your microscope test was done at higher solids See TechNotes 201 204 206 301 and 304 for more information DNA Adsorption Q read in the protocols of Whitehead Institute that for purifying single or double stranded DNA you use carboxyl particles How can you convince DNA to bind to negative charges Or do metal ions create DNA Purification suitable bridges Superparamagnetic Short answer Yes But you know we won t leave it at that
7. ssssssseeeenenennnnnnnnnenes 13 Quality Gontrol oorerrorerrorrrrorroroorororvnronvnrenvnrenenvensnn 17 19 Quantum Dots sss nnns 10 Quantum FITC MESF erererrrvovorovorvrrrvorererrsrereresesversvesesnene 10 Quantum MESF Kit 2 11 12 15 19 23 Quantum Simply Cellular 2 4 7 11 14 16 22 24 Quiet enne rtt etus 27 46 60 QUIGKGAI ii eee Ries ite 21 R Rare Earth Magnets sss Reaction Purification essen Reagent Stability o oeorrrnorrorrrrrorererrsrsvereresvsrsvonesrsvovener Recombinant Antibodies esse Redispersing Beads sse Red Laser Far red Detection 12 Reference Beads 18 References 6 15 18 Mone 21 22 23 25 26 WELL 28 30 31 39 42 44 E 46 54 57 60 61 63 Reference Standards 14 Refractive Indices 57 Regional Blood Flow Tracing 45 Replicate Lots 39 Replication Tips sese 39 RED KOCUGCIDINTY cett the eer rer rts 58 Reproducing Clump SizeS osorrvrnrorororrrroverevesvorsvenesrsvovener 50 Reverse Passive Latex Agglutination RPLA 45 S Sandwich Strip Test cccccsecscsecscsscscsseecssesees 54 55 58 63 Scattered Light Measurements
8. Bangs Laboratories e Ask The Particle Doctor Page 64 Aggregation Clumping Electrolytes Flocculation Hydrophilic vs Hydrophobic Surfaces Surfactants m having problems coating particles with protein The particles are stable when I start but when I add the protein solution they form visible clumps Sometimes there are problems with flocculation clumping of the small particles upon addition of protein buffer solution Often the buffer electrolyte is causing the particles to flocculate before they can become protein coated Most of our particles are made with fatty acid surfactants Added as emulsifiers during polymerization they act as colloidal stabilizers after polymerization The hydrophilic COOH groups and at basic pH s the charged COO groups from the adsorbed fatty acid molecules make the surface of the polystyrene particles more hydrophilic Permanent charged SO groups on the ends of the polystyrene polymer chains from free radical initiator used in polymerization also add hydrophilic character to the particles In pure deionized water particles covered with these negatively charged groups are mutually repulsive and therefore completely dispersed colloidally stable Nevertheless the particles still have exposed hydrophobic polystyrene surface Once the hydrophobic surfaces of adjacent particles come into contact with each other they will stick together firmly Electrolyte added to the water can decrease t
9. Long answer The folks at Whitehead in their Beads work on the Human Genome Project use 10 mM MgCl salt and 13 polyethylene glycol 8000 PEG to cause the DNA to bind to the surface of the COOH modified magnetic beads We believe that it is the divalent Mg which is causing the DNA to adsorb onto the magnetic microspheres which they then use to separate the DNA from cell debris Gravimetric Analysis Q How do you determine the solids content of your microspheres On a spectrophotometer Percent Solids Determination A No a spectrophotometric method to determine the solids content of microspheres is valid only when a standard curve is generated using the same batch of microspheres and when they are always handled in Spectrophotometer the same manner aggregated microspheres scatter differently than single microspheres and particles coated with protein scatter differently than non coated beads It is because of these drawbacks that we do not use spectrophotometric concentration measurement Our method is gravimetric and is we feel much more reliable Although it may require quite a bit more product we determine the dry weight of a specific weight of suspension Lectin Coupling Q I want to couple lectin to your magnetic beads Will get more lectin bound and better oriented if I covalently bind the lectin directly to the beads or if buy your streptavidin coated beads and hook on some Ligand Orientation biotinylated lectin Streptav
10. nd 4 7 85 10 um IgG in this case The surface area of a 0 8um microsphere A nD 2 01um 0 8um bead Therefore you can pack A a 2 01 7 85 e 10 25 600 IgG s bead The general solution is simply A a nD nd 4 4D d Or here A a 4 0 8 0 01 25 600 You can of course get different values by considering IgG s elongated or ellipsoid shape and possible orientation standing up or lying down Singer used a whip Q After coating my polystyrene particles with IgG I found that they were clumped Can you help me We may not be able to help with the already clumped and coated batch but we may be able to prevent this from happening again Examine the particles at every stage of the processing to ensure continual single particle suspension Look at the particles under a light microscope 1000X magnification oil immersion Normal appearance is a continuous sea of single particles swimming or vibrating rapidly under Brownian motion with occasional doublets or very few small clumps of dried particles knocked off the sides of the bottle during mixing You cannot see single particles lt 0 4um but you can detect the larger slowly moving clumps Ultrasonics will break up the loosely bonded clumps which result from centrifuging particles but will not remove tightly bound clumps resulting from freezing drying or chemical agglomeration If the particles do become clumped during processing you may remove very large clumps by
11. 4 x 10 IgG molecules on each microsphere Remember different proteins will have different affinities for a bead surface Also more isn t always better but depends on conformational changes and steric effects You must test to determine how much adsorption is needed for best performance Q What size amine microspheres should I try for coupling reactions with NHS esters Of course the devil in me suggests that you try all the sizes But seriously folks for a proper response we must ask you for more information What do you want to do with the microspheres What sort of assay or application do you have in mind The size of bead is typically dictated by the application or assay format etc Size will impact bead handling surface area area for immobilization of biomolecule settling times etc For example flow cytometric tests and assays typically make use of beads that are 2 8um strip tests typically require beads that are 0 1 0 4um Our 300 series of TechNotes describes a number of applications with usual bead sizes noted Also see TechNote 402 published article with a link to the publisher s website which contains recommendations on bead sizes for a number of formats Of course all our TechNotes may be downloaded from our website www bangslabs com Q I m looking for some beads to calibrate my cytometer in terms of GFP molecules heard that you made such a kit but can t find it on your website Although we previously p
12. 7 3 231 240 PubMed ID 7577826 Puela J M et al 1995 Coadsorption of IgG and BSA onto sulfonated polystyrene latex II Colloidal stability and immunoreactivity J Biomater Sci Polym Ed 7 3 241 251 PubMed ID 7577827 Zalazar F E et al 1992 Parameters affecting the adsorption of ligands to polyvinyl Bangs Laboratories e Ask The Particle Doctor Page 26 Adsorption Equations Ligand Bead Calculation Parking Area Surface Area Surface Monolayer NHS Esters Particle Determination Technical Literature Calibration GFP EGFP Coupling Efficiency Oligo Binding References chloride plates in enzyme immunoassays J Immunol Methods 152 1 1 7 PubMed ID 1640104 Q How many protein molecules can adsorb onto 1yum microspheres If we assume that you are packing them tightly like a monolayer then you should be able to calculate the number of protein molecules per bead as follows 1 From the Stokes diameter of the protein molecule you can calculate that a spherical protein molecule would occupy an area cast a shadow of md 4 If the diameter of IgG is 10nm then its parking spot on a microsphere would be 78 5 sq nm 2 The surface area of a microsphere is nD Then a 1um 1000nm microsphere has 3 14 x 105 sq nm or 314 x 10 sq nm of surface area 3 You can therefore expect to be able to pack a maximum of mD md 4 4 D d molecules per sphere In this case it would be 314 x 104 78 5
13. Limited These products are manufactured under license from Carnegie Mellon University under U S Patent Number 5 268 486 and related patents ICI Americas Inc Tween Molecular Probes Alexa Fluor Texas Red Pierce Biotechnology Inc Slide a Lyzer Polysciences Inc BioMag BioMag Plus Polysciences Inc ProMag SNARe ViaCheck Union Carbide Corporation Triton is a trademark of Union Carbide Corporation which is a wholly owned subsidiary of The Dow Chemical Company
14. Magnetic Beads Magnetic Separators SNARe Alexa Fluor 488 Anti Mouse IgG Beads LSR PMT 714 Glacial Blue 2 3 2 e so 2o os a 2 a 2 45 cs So og o a E 10 10 10 10 10 Pacific Blue A APC Cy A Catalog Code Description FC06F 6952 Glacial Blue Microspheres 914 APC Cy 7 Reference Standard 913 Far Out Red Microspheres am developing a bead based assay for use in our laboratory and other clinical research laboratories and could use some pointers on quality assurance and standardization Where can I find information Our new Flow Cytometry Supplement including a technical reference guide outlines a basic program for quality assurance and standardization in the flow cytometry laboratory and provides some references that pertain to the clinical research laboratory in particular Supplementavailable for download from our website The Clinical and Laboratory Standards Institute www nccls org and the International Society for Analytical Cytology ISAC www isac net org are excellent sources for information If you re not already a subscriber you might also sign up for the Purdue University Cytometry mailing list which is basically an email forum for all things cytometric You may submit questions regarding protocols products instrumentation regulatory issues etc which will be zealously addressed by flow ers from all over the world http www cyto purdue e
15. S0 it s lights out eh Sorry that wasn t punny at all Getting down to business Many fixatives mounting media and adhesives have components that act as solvents Organic solvents will swell the polymer matrix and allow release of fluorophore A water soluble mounting medium e g Mowiol Catalog 17951 Aqua Poly Mount Catalog 18606 Polysciences should resolve the problem In fact aqueous mounting media are used with fluorescent microspheres in the production of Polysciences Confocal Multifluorescent Adjustment and Calibration Kits Catalog 424016 Other alternatives include using surface labeled beads as in our flow cytometry line though these won t be as bright as internally dyed beads i e fluorescence intensities are nearer to those for stained biologic samples or beads synthesized using fluorescent monomer see our sister company s Polysciences Fluoresbrite PolyFluor Microspheres Please note however that the polymer base bead will be susceptible to the effects of solvent so there may be diminished signal just purchased carboxylated beads which have been coating with antibody Though I have little experience with this have had continued problems with reproducibility sometimes achieving good results and other times experiencing very low coupling What can do to improve my results Bangs Laboratories e Ask The Particle Doctor Page 7 PolyLink Protein Coupling Kit Affinity Interac
16. Storage Shelf Life Shelf Life COOH Modified Beads Particle Determination Spacers sticking together to form large clusters What happened Always make sure that any microspheres that you use are singly dispersed before and after dilution Look at them under a microscope Even the smallest ones are visible with a light microscope 100X objective oil immersion lens if they are clumped Silica beads are more hydrophilic but if they do become clumped you must ensure that they are well redispersed This may require ultrasonication See TechNote 202 Q Can I bind two or more different proteins to microspheres at the same time It should be possible to bind a small amount of one protein to the particles careful not to cover up all the surface followed by a second protein OR Mix and bind the two proteins together Some folks mix IgG and BSA or HSA in a certain ratio and bind them to give proper level of IgG on the microsphere surface with the albumin acting as blocker See TechNote 204 Q How important is the pH of the storage solution for the shelf life of the carboxylated latex beads A It is best to store latex in water with added surfactant Keep it fresh in the refrigerator Increasing ionic strength is bad for particles in general Best pH probably is slightly basic to convert COOH groups to COO groups Q What is the expected life of carboxylated beads A With no bacterial contamination beads will probably
17. and acid levels Also the chemistry has been well worked out and recipes published However we also have a few particles with other surface groups like amino and hydrazide which are also very useful for coupling Lateral Flow Test After we dry the Ab coated dyed particles onto our membrane how do we ensure that they will move Membranes when re wet with sample Membrane Pre Coating Lack of movement could be due to particles too large to move freely in the membrane particles sticking to the membrane or a sample flow rate inadequate to dislodge and carry the particles along the strip Mobility of Particles You could investigate the mobility of the particles through the membrane as follows Sticking 1 Spot some particles on the membrane 2 Before they can dry add water or buffer to move the particles Strip Tests 3 If they do not move then there may be a physical barrier to their movement Sticking on a bare membrane may be prevented by precoating the membrane with proteins Alternately or additionally the membrane surface can be treated with very hydrophilic materials like sucrose which should easily rehydrate and release the particles upon re wetting Other hydrophilic materials might also work such as trehalose other saccharides and oligosaccharides Gelman suggests using BSA non ionic surfactant and sucrose See TechNote 303 Lateral Flow Tests Bangs Laboratories e Ask The Particle Doctor Page 63 Lateral Flow Te
18. challenge the membrane with them Spheres that escape the membrane could be dissolved in a solvent for polystyrene to release the dye Then if enough dye is present a spectrophotometer or fluorometer could pick up the dye signal from those particles It may be possible to detect the particles if you could find them In the particles the dye would be concentrated enough to detect with a fluorescent microscope Q Tell me about the latex beads used in RPLA tests where latex beads are used to simulate red blood cells RBCs Microspheres for RPLA should behave like RBCs since RPLA was developed to mimic hemagglutination tests So they should be large dense enough to settle within about an hour or more depending on the time allowed for test results One can use large PS beads with density of 1 05 g mL and we can help you calculate the time for settling of various diameter PS beads TechNote 206 If you want smaller beads with higher surface specific area try PMMA At a density of 1 19 g mL PMMA beads will settle 4X as fast as same size PS Silica beads density 2 00 g mL can also be used these high speed beads will settle 19X as fast as the PS RPLA beads probably should be dyed to see the pattern in the bottom of the 96 well plate They don t need to be red but some dark color is probably best Q Protein in urine interferes in one of our urine based tests using your chloromethyl beads might prevent agglutination NSB ca
19. p density g mL or g cm Then for 1 micrometer 1 micron diameter polystyrene spheres A 3 1416um 3 1416 x 10 A sphere and V 0 5236um particle tom 10mm 10 um 10 nm 10 1cm 10 mm 10 um 10 nm 1075 1cm 103 mm 10 um 10 nm 1074 A Density of polystyrene is 1 05 g mL 1 05 g cm x 1 cm3 101 um 1 05 x 107 g um Then M 0 5236 x 1 05 x 10 0 54978 x 10 g particle Thus 1 M 1 819 x 10 charges g Then 6 02 x 1019 charges g 1 819 x 10 particles g 3 31 x 107 charges particle That s plenty Similarly you can calculate charges per particle for other sizes densities and surface charge densities The other conversion factors above will let you calculate Coulombs per particle from microeq g density and diameter We calculate and report the parking area or average area occupied by each charged group on the surface This parameter gives us a way to compare the amount of acid on the surfaces of different sized particles In the example above one sphere will have a surface area of 3 14 x 10 A and 3 31 x 10 charges particle or 31 4 3 31 9 5 COOH group COOH groups in a close packed monolayer of fatty acid each occupy an area of about 25 so the equivalent of much more than a monolayer of acid groups on the surface Do you recommend one step or two step covalent coupling to COOH modified microspheres A The short answer is Two step The inevitable
20. starting point from which to optimize Our PolyLink coupling kit features standard EDAC mediated coupling chemistry and includes a good general protocol see Product Data Sheet 4644 Q I ll be coating your 5 5um Protein A coated beads with IgG but would prefer to forego the crosslinking step with DMP Do you think that the IgG coating will be stable without the crosslinking Can count on the coating stability to be like that of covalently bound protein The affinity of protein A for IgG varies by antibody host species and subclass see a chart in TechNote 101 This means that without crosslinking the beads should be used in an environment that is otherwise antibody free As an affinity interaction it may be susceptible to competitive binding dissociation of the intended antibody through competition with Abs in the sample You will also want to consider the inherent stability requirements ofthe application in addition to the desired shelf life For example for quantitative assays extended stability or if target is to be eluted would suggest crosslinking If the beads simply need to capture target for a qualitative application and will be used to fulfill a short term objective i e a lengthy shelf life isn t required then crosslinking may not be so important purchased your Quantum Simply Cellular amp beads to evaluate surface marker expression However I m new to this product and flow cytometry in general and am a bit ne
21. too much BSA might cause competition between BSA and IgG molecules 2 Level of IgG coating perhaps using a higher concentration of protein will allow you to load more on the surface making it less accessible to BSA molecules in the buffer 3 Use of BSA as a blocker other blockers might not compete as efficiently for the surface 4 Use of PBS huffer other buffers might provide improved storage stability Another factor to consider is that proteins tend to become more tightly adsorbed to surfaces over time The loss of activity that you have observed may be caused by this phenomenon as molecules become more tightly adsorbed they undergo conformational changes that can reduce activity If this is the case using a higher IgG concentration might improve loading and force molecules into a crowded upright position To investigate you might utilize a total protein assay to determine if the protein level is remaining constant on the beads or in the supernatant If it remains constant this might indicate competitive desorption replacement of IgG molecules with BSA molecules or loss of activity due to conformational changes of protein See our TechNote 205 Covalent Coupling for references and suggestions on assaying beads for protein load and activity Here are some other references Puela J M et al 1995 Coadsorption of IgG and BSA onto sulfonated polystyrene latex I Sequential and competitive isotherms J Biomater Sci Polym Ed
22. 1 5mL Magnetic Separator MS002 BioMag Multi 6 Microcentrifuge Tube Separator MS003 BioMag 96 Well Plate Separator MS004 BioMag Flask Separator I see that you sell standards for some but not all of the fluorochromes that use For some Cy Chrome BD Biosciences simply need a reference standard and for others Alexa Fluor 488 Molecular Probes Inc need to be able Bangs Laboratories e Ask The Particle Doctor Page 14 Cy Chrome Fluorescence Quantitation Fluorescent Reference Standards Protein A Microspheres Quantum Simply Cellular Reference Standards Carboxylated Beads Directed Immobilization EDAC Coupling PolyLink Protein Coupling Kit BioMag BioMag Plus COMPEL Flow Cytometric Assays Magnetization Purifying Samples to quantitate the fluorescence signal What can use For fluorescence quantitation the same fluorophore must be on beads and cells This ensures that the beads respond to the environment in the same fashion as cells and quantitative assignments are truly relevant Our Quantum Simply Cellular kits are comprised of bead populations labeled with anti mouse anti rat or anti human antibodies They may be labeled directly with your fluorochrome conjugated primary antibody or indirectly using your unlabeled primary and a labeled secondary See our online Flow Cytometry catalog for Product Data Sheets For a simple reference standard the
23. 1996 Reversibility of biotin binding by selective modification of tyrosine in avidin Biochem J 316 Pt 1 193 199 PubMed ID 8645205 Also the prebinding of biotin before binding of biotinylated ligand has been cited much more extensively i e it is the basis of several competitive assays This may help control the level of loading when using streptavidin coated beads Q What is quenching Is it the same as photobleaching Quenching and photobleaching are two different phenomena Quenching is the loss of fluorescence intensity due to interference between fluorochromes It usually occurs when fluorochromes are closer together than 50 angstroms too many dye molecules per bead Quenching is a reversible proximity issue Photobleaching on the other hand is an actual alteration or destruction of the fluorochrome by the excitation energy or ambient light like your favorite shirt that just fades in sunlight That said if you can find a way of quenching photobleaching please let us know What do you know about the stability of the streptavidin biotin complex vs pH After binding of SA B I want to do a reaction at acidic pH 3 5 Bangs Laboratories e Ask The Particle Doctor Page 28 References Streptavidin Biotin IgG Binding Latex Agglutination Tests Brightness MESF Values Adsorption Affinity Binding Coating Beads Covalent Coupling A You should be OK The book Avidin Biotin Chemistry A Handbook n
24. 700nm absorbance with these coated beads yet saw agglutination under a microscope when I put 5pL of 2 5 beads on a slide and added 5uL of 800ug antigen mL Lower concentrations were ineffective The specific surface area of your beads is 6 0 25 x 1 05 23 m g of beads So you could adsorb 23 x 3 69mg IgG g beads maximum adsorption rate of IgG onto polystyrene is 2 3 mg m If you used 250uL of 10 solids 0 025g of beads it would take 69 x 0 0259 or 1 7mg of IgG to coat the beads with a monolayer of protein You added 2mg IgG So there would be little extra protein to achieve equilibrium concentration in solution If you used 250pL of 10 solids latex coated at 1 solids then you had 2500uL of coating solution You added 2mg IgG In coating the beads you bound 0 8mg IgG so there must have been 1 2mg IgG in 2500uL of equilibrium aqueous solution or 1 2mg IgG 2 5mL 0 48mg IgG mL Compared to data from The Latex Course this is normal performance You put 0 8mg IgG onto 0 025g x 23 m g or 0 575 m of particles this is 0 8mg 0 575 m 1 4 mg m Published data would predict a monolayer at 2 7 mg m depending on pH at an equilibrium concentration of 0 5mg IgG mL You should get best orientation Fc portion down and maximum packing around pH 7 8 for rabbit IgG If the coating pH is very different from the assay pH you may lose protein as it changes conformation on the surface Also you may not want a monolayer of IgG for
25. Beads Covalent Coupling Equilibrium Adsorption Maximum Adsorption Rate IgG Monolayer Protein Loading Custom Coupling DNA Attachment ProActive Silica Microspheres 1 The particles are stabilized with negative surface charge sulfate groups from the emulsion polymerization initiator sulfonate or sulfate groups on the surfactant used to emulsify the monomers and the COOH groups from the vinyl carboxylic acid comonomer added with the styrene Even if all the surfactant is removed as by washing the particles should be stable in deionized water from the S04 and COO surface groups If necessary you can add a very small amount 0 001 0 01 of nonionic surfactant like Tween 20 or Triton X 100 to further assist in stabilizing particles Keep surfactant concentration as low as possible so as not to interfere with protein binding Keep the ionic strength of all solutions in contact with the particles as low as possible to avoid aggregation Keep the particles as dilute as possible to minimize chance of aggregation Dyed particles may have less surfactant after the dyeing process and the attendant clean up to remove dye from the aqueous phase You did not mention how you were coupling your protein so we will assume water soluble carbodiimide WSC coupling Adding protein and WSC should not destabilize the particles If you add protein first it should adsorb on and assist in stabilizing the particles Also you may have better luc
26. Cellular anti Mouse Compensation Standard Do you offer anything forthe direct binding of goat hamster or rabbit antibodies Though we don t offer any anti goat anti hamster or anti rabbit IgG standards per se we now offer single population Protein A and Protein G microspheres that are suitable for binding a range of antibodies and may be used as reference beads for flow cytometry Protein G is a strong binder of goat and rabbit antibodies and Protein A binds hamster antibodies You may use an unlabeled population or our Certified Blank microspheres with the labeled population for compensation purposes Cat Z Description 553 Flow Cytometry Protein A Antibody Binding Beads 554 Flow Cytometry Protein G Antibody Binding Beads 890 Certified Blank Reference E re E FTCA Protein A beads labeled with IgG FITC Standard Bangs Laboratories e Ask The Particle Doctor Page 5 Aggregation Bangs Bead Buffers Bangs Bead Solution Charge Groups Hydrophobicity Polystyrene Microspheres Surfactants Coupling Strategies Lipopolysaccharides Flow Cytometry Quick question How does Bangs Bead Solution impact subsequent coating for example will need to do a lot of washing to remove it before coating Will beads aggregate after washing The Bangs Bead Solution SOLN1 is a good dilution and storage solution for uncoated polymer and magnetic beads It contains an antimicrobial and stabilizers an
27. Passive Latex Agglutination RPLA Visibly Dyed Beads Agglutination Blockers Ghloromethyl Beads COOH Modified Beads Nonspecific Binding Quenching I have been using 2 and 5 micron beads in an immunoassay where the antibody on the bead needs to bind to a surface bound antigen I do get adsorption of the beads to the surface but no significant binding Is it that the beads are too big and won t attach well to a smooth surface A If your 2 and 5um beads are coming off the smooth surface it is possible that they aren t bound tightly not enough Ab on the surface Ab binding sites might not be directed outward or perhaps rinsing or washing following staining is knocking the large beads off the surface For the first two possibilities you could try other binding methods If you suspect the latter then we offer smaller particles which would sit closer to the smooth surface and be less likely to be removed by any vigorous washing Weare testing a membrane that captures 20nm particles As part of the membrane challenge the membrane should demonstrate a 5 log 10 000x reduction efficiency That is for every 10 000 particles put on the membrane only 1 gets through Do you know of any way to accomplish this challenge How do you detect one or two 20nm particles coming through the membrane Radioactivity has been ruled out for now A One method might be to heavily load 20nm microspheres with a fluorescent dye 10 20 dye and
28. Small Microspheres With the help of Microgon we recently did a double exchange of some 0 1 um beads Using a 0 05um polysulfone hollow fiber membrane we changed our PS and PMMA beads from water to ethanol it sure smelled good to a halogenated solvent which was nota solvent for PS Organic Solvents What about other polymers like polymethyl methacrylate PMMA What kind of organic solvents will dissolve PMMA PMMA We do not have a list of solvents non solvents for PMMA but we expect that the list will be similar to that References for PS You can get this kind of information from books on solubility parameters such as Handbook of Polymer Liquid Interaction Parameters and Solubility Parameters by AFM Barton CRC Press 1990 Aerosolization Q How about changing phase from water to air drying the beads Changing Phases Drying the larger beads is easy Let them settle out of suspension and decant the water or change phase Drying Beads to alcohol faster settling and faster drying then let them dry and break up the pellet with a spatula stir rod or even a mortar and pestle the latter works well for our silica beads The smaller beads will settle slower and will form a harder pellet In some cases it may be easier to aerosolize the beads The idea is to disperse them in a liquid water or alcohol in dilute form then spray the suspension into a bag or other container suitable for collection If diluted properly each droplet will h
29. amine reactive dyes that are often used in flow i e for user created reference compensation standards see Amines to an End July 2008 e Silica has been used to support lipid bilayers in the creation of artificial cells to study things like membrane receptor ligand dynamics via flow cytometry and for specialized biosensing applications For specific examples see Lauer S B Goldstein R L Nolan J P Nolan 2002 Analysis of cholera toxin ganglioside interactions by flow cytometry Biochemistry 41 6 1742 1751 Zeineldin R M E Piyasena T S Bergstedt L A Sklar D Whitten G P Lopez 2006 Superquenching as a detector for microsphere based flow cytometric assays Cytometry A 69 5 335 41 e Silica microspheres have proven to be an excellent support for oligonucleotides in hybridization based assays The silica surface is negatively charged which is helpful for deterring the nonspecific binding of DNA Silica is also highly hydrophilic and nonspecific binding of proteins which largely relies on hydrophobic interactions is less than that seen with many polymer based beads These are just some of the silica microsphere applications that have made an appearance in flow cytometry and we re sure that we ll continue to see more as investigators explore its unique properties Q I m having trouble seeing my fluorescent beads after mounting them on a slide Any ideas as to why this is or what can do to prevent it A
30. amount of surfactant will probably not cause much desorption but to be safe we would recommend covalent binding What is the best way to couple a decapeptide firmly to a bead without losing activity of the peptide Others coupled the peptide to IgG via cysteine using succinimidyl 3 2 pyridyldithio propionate and then adsorbed the IgG to the latex bead See S Miyamoto et al 1995 Science 267 883 885 especially note 4 p 885 We put a cysteine on the end of our peptide so we can it this way but could we couple the peptide more directly to the bead We expect that you care whether you can put more active polypeptides on a bead surface by direct tail down covalent binding OR by binding polypeptides to IgG followed by adsorption of the IgG onto a polystyrene bead We suppose that you can put more peptide on the particles with IgG but we don t know how much will be active You also have the option of binding your peptides to IgG or to beads using spacers of various lengths to increase activity Yes You can certainly bind your polypeptide to any of several surface functional groups Popular groups are COOH NH hydrazide epoxy CHO hydroxyl and chloromethyl diluted a 10 suspension of your 1 6um diameter silica microspheres with water resulting in a solution of 1 solids However most of the beads were Bangs Laboratories e Ask The Particle Doctor Page 47 Silica Microspheres Blockers BSA Protein Coating
31. anti Human Catalog Code 812 IgG surface When stained the beads will bind a known number of your fluorescently conjugated mouse or human IgG antibodies QC of the antibody is as simple as monitoring the fluorescence intensity of the stained beads When used in conjunction with one of our Quantum MESF kits the Simply Cellular beads will allow you to determine the effective fluorochrome protein F P ratio of your antibody SSC H 200 400 600 800 1000 0 0 200 400 600 800 1000 FL1 H Bangs Laboratories e Ask The Particle Doctor Page 20 BioMag Q would be interested in using biotin coated beads Do you have them available Biotin We certainly do We offer BioMag biotin coated superparamagnetic particles as a standard product We also have custom coating services and would be pleased to coat a base bead selected from our many polymer magnetic fluorescent or dyed microsphere product lines Crosslinking Q would like to bind a peptide to microspheres What type of microsphere do you recommend Functionalized Microspheres A For peptides and other small molecules you may wish to employ the use of a spacing molecule to ameliorate steric effects or a crosslinker to target a specific residue and optimally orient the molecule Peptide Binding Crosslinking agents are available with a variety of reactive groups for use with functionalized microspheres covalent coupling or with a biotin molecule for affinity binding to
32. assumptions 1 Colored line must be 5mm long x 0 5mm wide x 10 particles deep or thick 2 The particles are 0 25um diameter It will take 10 molecules to hold each particle at the strip location Binding will be 10 efficient there should be a 10X excess of molecules present to get proper binding Antigen molecules might be 1000 10 000 MW C1 RD TO IO wa Thus there will be 20 000 x 2 000 x 10 2 4 x 10 particles and 4 x 10 molecules for binding the beads and 4 x 101 molecules or 67fM 67 667 picogram sensitivity is possible Please ask for a more complete explanation if you wish Last year bought some 0 300um particles How close can you get to 0 300um again with an entirely new lot We can usually reproduce lots to 5 of the original size or within the range of 0 285 0 315um Often we can get closer We can usually show you two or three replicate lots where we tried to repeat a given recipe several times Please try other sizes since most people find no difference between particles of 20 from the original size 0 24 0 36um I want to replace enzymes and some steps by using dyed particles in a sandwich test format on coated tubes What size particles should use Try the smallest particles 100nm which can move fast and stick tightly to the tube surface If larger particles say 1m were used they might stick out from the surface too far and be more likely to be knocked off by liquid swirling arou
33. at pH gt 3 7 So at any pH gt 4 where your DNA or fragment is positively charged DNA should adsorb onto SiO It is also possible to reverse the silica charge and adsorb negatively charged DNA See Bangs Laboratories e Ask The Particle Doctor Page 57 Light Scattering Assays Nephelometric Assays References Refractive Indices Surface Tension Surfactant Concentration Parking Area Titration Data Equations Parking Area Surface Area Titration Data TechNote 104 for more information 2 Polymeric particles e g plain or magnetic polystyrene will also adsorb or covalently couple streptavidin The streptavidin will bind biotinylated DNA or oligonucleotide very securely See Product Data Sheet 714 for the protocol 3 Amino terminated oligos will bind to COOH modified particles via water soluble carbodiimide See TechNote 304 for the protocol Q Could make better nephelometric assays using particles with refractive indices higher than polystyrene s There is interest in particles with refractive indices different from that of polystyrene both higher and lower refractive indices have been the subject of recent papers One group showed that higher refractive index polymers can be made using vinylnaphthalene as the core and a shell polymer with active surface groups for covalent coupling These particles are better light scatterers especially after agglutination Deleo D T I R Lee J D Wethera
34. both kits include five bead populations one blank and four populations labeled with increasing amounts of fluorophore MESF or antibody QSC calibrated in terms of their Antibody Binding Capacity ABC The MESF beads are run as is the QSC beads are labeled with the same antibody that is used to stain cells The Bangs Laboratories e Ask The Particle Doctor Page 11 Quantum Simply Cellular Simply Cellular Staining Samples High Throughput Format ProMag fluorochrome labeled microspheres are run onthe cytometeratthe 181 000 e same instrument settings as cells Their channel values are then 9500 used to generate a standard curve relating fluorescence intensity 34000 e to standardized MESF or ABC values from the beads The MESF 8000 e or ABC values of the labeled cell samples may be determined by measuring their fluorescence intensities and reading the Event Count corresponding MESF or ABC values from the standard curve using Reference Blank the QuickCal analysis template that is provided with the kit s Some differences are as follows J S Relative Channel Number J Quantum MESF e Kits are available in FITC PE PE Cy 5 and APC versions e Prelabeled beads are very convenient to use and are not subject to the same variation that could be introduced through staining different technicians antibody lots etc e There is no mAb consumed to stain the
35. cause it to resist nonspecific References binding of negatively charged molecules such as nucleic acids However nothing is absolute adsorption is dependent upon a number of factors including characteristics of both the biomolecule concentration solubility Silica Applications charge pl etc and the solution pH salt content presence of competing molecules etc In fact our silica microspheres have been utilized for the study of protein adsorption as reported in Silica Microspheres Docoslis A L A Ruskinski R F Giese C J van Oss 2001 Kinetics and interaction constants of protein adsorption Volume 16 2 onto mineral microparticles measurement of the constants at the onset of hysteresis Colloids and Surfaces June 2003 B Biointerfaces 22 267 283 van Oss C J A Docoslis R F Giese 2001 Free energies of protein adsorption onto mineral particles from the initial encounter to the onset of hysteresis Colloids and Surfaces B Biointerfaces 22 285 300 ESS Our silica microspheres have been proven to be useful for many applications including the following e In solid phase diagnostics after functionalization of the surface for coupling of biomolecules e For nonspecific immobilization or purification of nucleic acids in the presence of divalent cations or in the presence of a chaotropic agent and high salt NW SI an Bangs Laboratories e Ask The Particle Doctor Page 23 e As substrates for t
36. concentrations Low Particle Concentration We always measure solids content by loss on drying weigh out 200uL of particle suspension evaporate water weigh solids remaining and calculate solids suspension ratio 9o solids This method would be quite Scattered Light difficult for very low lt lt 1 solids Measurements You can try to measure solids content turbidimetrically with a spectrophotometer if you understand that light Solids Content scattered by the particles is a function of the concentration of particles the parameter you want to measure Scattered light is also a strong function of several parameters which you do not want to measure like particle diameter particle aggregation singly dispersed particles vs clumped particles polymer refractive index particle coating may affect refractive index and wavelength of light used You can be successful measuring solids if you are very careful to observe the following precautions 1 Carefully prepare serial dilutions of particles with surfactant solution to ensure complete single particle dispersion Measure and plot absorbance vs concentration 2 Measure your unknown under exactly the same conditions 3 Coated particles may behave differently from clean bare particles 4 Prepare a fresh curve for each different particle type and diameter Cross Flow Filtration Q What s the difference between ion exchange IX and cross flow XF filtration cleaning of particles lo
37. could share with me Firstly don t despair a couple of common issues come to mind and they re generally easy to fix As a first step well second step if we count the not despairing part you should ensure that the beads are being washed sufficiently prior to coating The as supplied storage buffer contains a blocking molecule and other stabilizers that could reduce binding efficiency As a matter of course we suggest a few pre washes 3X centrifuge decant resuspend in buffer to remove these prior to coating with the biotinylated ligand You will also wantto ensure that your binding buffer is free of or contains only minimal amounts of potential interferents such as blocker surfactant etc I ll also note that we use a biotin FITC assay to determine binding capacity As a small conjugate 831 Da biotin FITC is able to efficiently access streptavidin binding pockets The capacity of the beads for biotinylated protein will be somewhat subject to steric effects i e as we expect the far larger protein to mask binding sites that would be accessible to small molecules Using the Surface Saturation equation that is provided in our TechNote 206 will probably give you a better estimate of the amount of protein that can be coated onto the surface and adding some amount more than this will aid in achieving saturation If your protein is of gargantuan proportion I m thinking IgM scale you might consider biotinylating it with a reagent t
38. currently qualitative only but need to ensure that the changes I am seeing are not just relative but quantitatively different thought that using beads and adding a known concentration to my sample would allow me to determine the absolute number of white cells that have Basically want to take a blood sample add the beads lyse the red blood cells and then use the flow cytometer to quantify the WBC concentration Could you recommend beads that could use for this process The less expensive the better We have just the product for you It is called Flow Cytometry Absolute Count Standard The beads are roughly 7 8 micron in size and exhibit broad spectrum fluorescence Just as you mentioned beads are added to your cells prior to acquiring them on the cytometer and using a simple equation provided in the Product Data Sheet allow you to calculate the concentration and absolute number of cells in the sample Catalog Code Description 580 Flow Cytometry Absolute Count Standard perform standard WBC immunophenotyping and use your fluorescent beads as controls I m worried that differences in my conjugated antibodies either lot to lot or over time may be contributing variability to my results Is there any way to QC my antibodies using the bead controls OG of fluorescently conjugated antibodies may be performed with our Simply Cellular microspheres The single population of beads has an anti Mouse Catalog Code 810 or
39. esse OG TAO accrescere P tr bate ea etes Catalog Code Meanings sene Cell D amp platiori rette ttes Cell Quantification sse Cell S parations 2 trece ee eet Centrifugation ssssssesene Certified Blank M cii te ere metet Changing Phases c cccccccccsescsescsscsescseseeecscsesesesecsssesesaeees Changing Solvents eee etess Characteristics essere Charge Groups x eter tete e metres Chemiluminescent based ELISA ssss Chloromethyl Beads Chloromethyl Binding oseervrorrvererrrnsversvorrsvovovererrnvsnevenenne Cleaning Methods sse Cleavable Crosslinkers seen Gl avable Linkers ttes Clumping oevevrrnrvorererrorsvoresrsvrvorerrsvsreverenn CML Binding eret tre e eee etie Co Adsorption esses esee nennen nnns Coated Microspheres esee Coating Bead Concentrations sss Coating Beads sssssssssee Common Window of Analysis sse COMPELEM temet idees Compensation sess Competitive Detachment oeoeerrnrvervvrvrvrrvererrrrorererenn Concanavalin A orerosnsvsvovorrrrsvrvereresvsverovesrsvsnovereresneneverenn Conformational Changes seoranererrrvsvorovosvsvsvoverevrrvereverenn Gontamination
40. full face mask elbow length rubber or plastic gloves and boots Organic Beads Chloromethy Binding Stahility Storage Active Ester Quenching Aerosolization Covalent Coupling Cross Flow Filtration Drying Beads Ethanolamine Freeze Dry Tween 20 2 If you just want to make hydrophilic beads then you could simply mix beads with ordinary concentrated nitric acid which is only saturated a bit easier to work with but not as concentrated as fuming red nitric acid which is supersaturated in NO The nitric acid will etch the surfaces and create hydrophilic groups like OH or COOH groups but it probably won t put nitro groups on the surface If you want to go this Way you can also forget about the glacial acetic acid in that recipe mentioned in my book For hydrophilic not nitrated beads you can just etch the beads for a few minutes then drain the beads by pouring off the nitric acid through a glass frit funnel Then rinse thoroughly with water do this very carefully since water added to acid will generate lots of heat and see if beads are hydrophilic enough If you place some on the surface of a beaker of water do they float hydrophobic or sink hydrophilic and do they cling together in water hydrophobic or are they water dispersible hydrophilic If you leave them in the acid too long they will dissolve since the acid just keeps on etching If you do this work with the help of a chemist this is highly recommen
41. functional group on the surface of our particles We titrate and report the amount of acid as microequivalents per gram of particles Q But what about the relative number of groups on the surface The specific surface area for a sphere m g is 6 d where d diameter in micrometers so 1um particles will have surface area 6 m g If you divide surface charge peq g by the specific surface area for that lot of particles you will get surface charge per unit area ueq m We invert this number and report the parking area A2 COOH group or area available per molecule of acid on the particle surface Keep in mind that a close packed monomolecular layer of acid groups would have a parking area of 20 25A7 COOH group Then you can see that particles with parking area 25 have a complete coating of acid groups while those with gt 250 would be only 10 covered with acid groups The parking area is a way to begin to select particles of different sizes with equivalent binding properties Bangs Laboratories e Ask The Particle Doctor Page 58 Lateral Flow Tests Sensitivity Strip Tests Particle Diameter Reproducibility Particle Determination Sandwich Test Coating Beads Surface Monolayer Agglutination Test Bacteria Hydrophilic Membranes Membrane Sticking Q What sensitivity is possible with chromatographic dyed particle migration strip tests A calculated the sensitivity from the following
42. next reference describes somewhat different findings 3 Gonzalez M et al 1997 Interaction of biotin with streptavidin Thermostability and conformational changes upon binding J Biol Chem 272 17 11288 11294 PubMed 9111033 Also Gonzalez M et al 1999 Extremely high thermal stability of streptavidin and avidin upon biotin binding Biomol Eng 16 1 4 67 72 PubMed 10796986 Biotin binding increases the midpoint temperature of thermal denaturation of streptavidin from 75 C unbound to 112 C at full saturation 4 biotin 1 streptavidin and for avidin from 83 C unbound to 117 C at full saturation i e that in both scenarios unbound and saturated avidin possesses greater thermal stability Again however deglycosylated avidin is expected to be less thermally stable than native avidin 4 We expect that 1 2 binding sites are available for binding on each molecule of strept avidin assuming that at least two sites are inaccessible due to immobilization of the molecule on the bead So biotin bound immobilized strept avidin 1 2X biotin should have stability intermediate between that of unbound Bangs Laboratories e Ask The Particle Doctor Page 24 COMPEL Uniform Magnetic Microspheres Volume 15 4 De cem GIN Anti Human IgG Antibody Binding Capacity Quantum Simply Cellular amp 0X and saturated 4X strept avidin One might speculate as to whether immobilization will confer further st
43. of the suspension i e for the presence of aggregates through microscopy Q Your TechNote 104 Silica Microspheres says it s possible to reverse the charge of silica from to to adsorb negatively charged DNA Do you have any references Well it turns out that Leigh Bangs who suggested rinsing clean silica in a 0 1 1 M CaCl solution hasn t been lying to us all these years Here is an actual reference Romanowski G et al 1991 Adsorption of plasmid DNA to mineral surfaces and protection against DNase App Environ Microbiol 57 4 1057 1061 Mg or Ca were 100X better than Na K or NH in the adsorption of plasmid DNA onto sand indicating a charge dependent process We re using some of your COOH modified microspheres and I just encountered the term parking area What s that Are you guys running a parking lot Yes a parking lot for molecules Actually the parking area permits one to compare particles with different titration values meq g or ueq g and different diameters for their surface charge density which relates to their relative stabilities and binding capacities for proteins Calculated parking areas A2 charge group are the reciprocal of the surface charge density groups A or groups nm and are calculated from the diameter and titration of surface charge of clean microspheres If the parking area for any lot of microspheres is 20 COOH group then the microspheres are assumed to be covered w
44. other reagents such as formamide nylon wool or ethanol Adsorption of WGA to non functionalized microspheres might also be considered A sample adsorption protocol is provided in TechNote 204 Bangs Laboratories e Ask The Particle Doctor Page 22 Yet another strategy would be to immobilize the lectin through affinity binding For example biotinylated WGA which is commercially available may be bound to streptavidin coated spheres While you may insist on coating the beads yourself you could also try our newest BioMag Plus product Wheat Germ Agglutinin Catalog Code BP530 Ready to go we ve done the work for you at least this step COMPEL I was thinking about trying your uniform COMPEL magnetic particles amp for flow cytometry What do you think 8 Flow Cytometry 8 A We certainly recommend COMPEL for flow cytometry The side scatter Be Volume 3 SSC vs forward scatter FSC plot demonstrates the three discrete September 2003 populations that COMPEL 3um 6um and 8um magnetic beads yield upon flow cytometric analysis COMPEL also exhibit low autofluorescence which is important for bead based flow assays Antibody Source Q I use your Quantum Simply Cellular amp kits to determine CD61 expression of platelets and their precursors The manufacturer of the anti CD61 PE I had been using is no longer offering that antibody F P Ratio If I switch to another antibody source will my results still
45. pH J Biochem Biophys Methods 47 221 231 describes use of Molecular Probe s OliGreen for DNA quantitation suitable for sSDNA and oligonucleotides 1 When coupling to a COOH surface in a two step carbodiimide EDAC reaction where step 1 EDAC activation of beads in MES pH 4 5 and step 2 Protein coupling in Carb bicarb pH 9 6 with excess protein is there really a need to block the unreacted sites with ethanolamine 2 Can a similar two step EDAC reaction scheme be used with the encapsulated magnetic particles have a protocol that shows diethanolamine at pH 10 5 Is diethanolamine pH 10 5 preferred 3 What is the surfactant used with the COOH beads Will this interfere with the coupling reaction Is pre washing recommended I know there are really seven questions not a couple as I bunched a few together But I figured if I worded it 1 7 then by the time you got to 77 you might have inadvertently hit the delete button Thanks for your help 1 Some protocols call for blocking of unreacted sites others omit it since the reactive intermediary will hydrolyze back to COOH anyway You may wish to determine empirically the need for quenching e g the impact on nonspecific binding 2 A two step protocol may be used for magnetic particles The reactive group particle size and MW of biomolecule will be more important than the composition of the base particle when deciding between one and two step protocols For example particle
46. pH 8 Clean to remove unbound protein Two Step Coupling Under these conditions each part of the reaction occurs at its optimum pH In the first step the carbodiimide is protonated to make it more susceptible to nucleophilic attack by surface COO groups In the second part the amino groups on the protein are in the NH not NH form Adsorption To make a diagnostic test should I adsorb protein on polystyrene PS or couple it to COOH modified particles Antibody Immobilization Simple adsorption works fine to put whole polyclonal IgG molecules on particles for agglutination tests You might need covalent binding if you want to put on IgG pieces Fab portion for example if you are COOH Modified Beads working with monoclonal antibodies they may not adsorb as well as polyclonals if you are working with our itty bitty 100nm PS particles which may not be as stable as COOH particles or if you have a supersensitive Covalent Coupling assay where a minute amount of ligand coming off the particles will interfere You probably will need covalent coupling if you are trying to put on small molecules like antigens peptides pieces of DNA or RNA or haptens They either won t adsorb on the particles or stay very long There are about a dozen different coupling chemistries you could use to bind things onto particles Covalent coupling to COOH modified particles is easier because there is a better choice of particles sizes types colors
47. pass through the membrane or 3 they were stuck to the membrane by hydrophobic bonding You probably got chromatographic movement because you dissolved the beads with the ethyl acetate and the dye moved up the strip Try smaller beads another membrane or some surfactant to better disperse the beads or block the membrane with protein and surfactant to make it less sticky for protein coated beads See TechNotes 301 and 303 Q How do we know when the particles are clean That is how do we know we ve removed the surfactant Microspheres are clean enough when enough surfactant has been removed so that protein coupling proceeds really well Really it s not far from the truth You want to remove surfactant until coupling is uninhibited and reproducible while the microspheres are singly dispersed After cleaning some folks actually add back surfactant under their own control to assist single microsphere coupling Most coating protocols call for 3 washes before coating It also depends on how the microspheres are washed continuously by cross flow filtration or batch process in a centrifuge and how much supernatant is removed each time this is related to how much water is left with the microspheres after each wash One can measure surfactant or protein coming off the microspheres with instrumental methods including surface tension or protein analysis to see when the wash water is clean Remember that you really need two good clean up me
48. settling or with a fine mesh sieve cloth 400 mesh sieve will remove gt 37um particles or clumps Did you wash the particles before protein coating and if so what method did you use If you centrifuged did you ensure that the particles were completely resuspended before going on to the next step of your protocol Small 0 5um microspheres are more difficult to spin down and to resuspend Try cross flow filtration for cleaning particles without clumping them ask us for details Many customers use this system and report good results with it When coating the particles with protein did you calculate the amount of protein required to coat these small particles One gram of particles will adsorb 15 p d mg of IgG where p density of the particles 1 05 g cm for polystyrene and d diameter of the particles in um Therefore you will need 57mg of IgG to coat 1 gram of 0 25um particles plus some excess to ensure complete coverage We recommend that you put the clean particles directly into the protein solution putting them into buffer first may cause clumping due to the ionic strength of the buffer After cleaning you want to get them coated with protein quickly After microspheres become coated with protein they will be more resistant to ionic strength clumping Did you use a blocker You may want to mix blocker and IgG and adsorb them together in one step avoiding separate adsorption and cleaning steps Surface area mass should also be
49. size will in part dictate the wash method that is utilized and whether washes can be accomplished relatively quickly before the reactive intermediary hydrolyzes 3 Our TechNote 205 has a more typical but generic COOH bead EDAC MES buffer procedure You might have better luck this way with some optimization as the optimal pH for an EDAC reaction is considerably lower i e 4 5 7 5 Also an amine buffer ethanolamine might interfere with the reaction between bead COOH and ligand NH 4 The surfactant utilized for the synthesis of COOH functionalized microspheres may vary by lot As this is something that is generally held as a trade secret by manufacturers we can t advise with certainty on the specific surfactant utilized or its concentration 5 We highly recommend the pre washing of microspheres If use of surfactant is indicated i e if beads are aggregating or clinging to pipette tips and tubes we generally recommend using the lowest possible concentration of a non ionic surfactant such as Tween 20 0 01 OK I bound SA to your beads and it really grabs the biotinylated thingamabob that wanted to bind but enough already want to recover my Biotin Thing conjugate Make the SA let go It s hanging on like a 3 year old kid A am glad to learn that you got good binding Also I located a reference that cited use of biotin to competitively detach biotinylated molecules from modified avidin Morag E E A Bayer M Wilchek
50. sss 49 Streptavidin esses tnn tnnt 23 Streptavidin Coated Microspheres 3 12 20 27 29 30 38 Streptavidin Biotin soeoernrornrorverervererverornerennnr 28 36 37 Streptavidin Biotinylated DNA esesess 57 Jig TEStS RENE 51 54 58 59 63 SUNE 49 SUlf tiC AGI suverene 41 Superparamagnetic Beads esses 36 Surface Area ssssssseeeennnes 26 51 52 58 Surface Charge Density sse 48 Surface Marker Expression ccccccccsessscsssseescsssececseseececaees 7 Surface Monolayer s overervorrnrorvrrorrorervrronrreronrerenvevennnn 26 58 Surface Tension essent teens 57 Surface Titration Value sss 31 Surfactant Free essen 38 Surfactant Concentration see 57 Surfactant Removal eesvrvvevvrververververververvesvesvesvevresessenvenrer 43 Surfactants ieren 5 27 46 50 59 64 Surfactants for Protein 56 Suspending Dry Microspheres ssrsnarsvorrenerrrverrrverrrnenn 31 Suspension Dispersity resnrvrrnrvrrrerrsvorervarrererrrrerrrvernrnennr 34 T Technical Literature 26 Technical Services 53 Thiol Activated Particles seen 54 Thiol Preservation 32 Titration Data sssse 38 48 57 58 TOWEN E Ex anvende ini 44 Troubleshooting Tips sn 7 Turbidimetric Ne
51. streptavidin coated microspheres Depending Spacers on reactive groups that are present on the peptide you may wish to first modify the microspheres to avoid peptide crosslinking If a homobifunctional linker is used like glutaraldehyde you will want to use it in excess Streptavidin Coated to prevent crosslinking or hairpin binding Microspheres Compensation Q What is compensation and why is correct compensation important Flow Cytometry Flow cytometers are designed to have a primary detector for each fluorochrome label e g FL1 FITC FL2 PE FL3 Cy5 etc Fluorescent signals emitted by fluorochromes can bleed or overlap into the secondary fluorescence detectors In order to remove this overlap the proper amount of signal must be subtracted from the secondary detector as a percentage of fluorescence intensity measured in the primary detector This subtraction is performed by the electrical circuits prior to collecting sample data or by software when analyzing the list mode files When the mean fluorescence of two populations of labeled standards are adjusted such that they have Fluorescence carryover C is the region of equal intensities in the secondary fluorescence detectors then the data bd UAI from the samples will be accurately compensated Fluorescence Wavelength nm Compensation What s the difference between the two compensation kits that you offer Is there a way can check to see if my instrument s com
52. surface chemistry If you need large microspheres for proper sensitivity of an agglutination test or to be caught on a filter of a certain porosity we may be able to supply these Then we will help you to be sure that they remain singly dispersed as you work with them and formulate your final product Alternatively if you want a 300nm microsphere product which will never settle we ll be able to guarantee it I still have problems adsorbing protein onto polystyrene microspheres without clumping them Have you any new ideas for coating particles painlessly This common problem often starts when you try to clean the particles by washing especially with a buffer Even if you use cross flow filtration you can clump some low surface charge polystyrene microspheres When you clean the microspheres or take off their sweaters Don t be afraid it s only a very clever metaphor you are removing the surfactant used to make and store them They may not be stable even in DI water In a hostile environment they may huddle together without their sweaters and even less stable in buffers any buffer can be a problem After you get them coated with protein put their overcoats on they will become very stable warm again You have several options 1 Don tclean the microspheres Just add your protein Put on the overcoat while removing the sweater The high molecular weight protein should have a higher affinity for the PS surface than t
53. the rest ends up as water soluble polymer WSP or polymer with so much acid that the chains are fully water soluble and they completely escape Bangs Laboratories e Ask The Particle Doctor Page 32 Coating Beads Cross Flow Filtration Isopropanol Binding Protein Covalent Coupling Magnetic Beads ProActive Thiol Preservation BioMag Plus Concanavalin A Coating Beads Concanavalin A Aggregation Covalent Coupling Non lonic Surfactant Small COOH Modified Beads the particle surface WSP is removed by ion exchange cleaning of the particles before they are titrated Of course some acid may also be buried in the interior of the particles and not titrated We report titration values ueq of COOH g and then calculate apparent parking area and report it for most lots of these beads For more on this topic see TechNotes 201 and 206 We could deposit our special hydrophobic coating on your beads from isopropanol Would that be okay for polystyrene If so are there other ways to get the microspheres out of aqueous solution besides centrifuging And finally since can work with large sized spheres what would be an ideal size to remove water and then deposit our coating 1 Isopropanol would be fine for the beads And you could add it to the beads directly because it mixes with water 2 Cross flow filtration is a good way to remove some but not all solvent water or isopropanol 3 If you used gt 1pm bea
54. well dispersed after prolonged storage roll the suspension for several hours perhaps overnight Monodispersity may be evaluated microscopically or via an automated particle sizer If aggregation is observed it can often be successfully treated with the addition of surfactant see TechNote 202 Microsphere Aggregation A periodic re evaluation of your stored material using the same processes and criteria that you use for qualification of new shipments should provide the needed confidence for long term storage and use If you do encounter a problem e g contamination aggregation etc it should be possible to re work and re qualify the material I m building a particle sizer and need to obtain calibration standards What products are available Accurate particle size and distribution analysis is critical to particle based technologies in industry and research The particle sizing instruments used to support research manufacturing and QC efforts in these sectors must be rigorously calibrated and validated to ensure the integrity of results We offer a full range of polystyrene based NIST traceable particle size standards that are suitable for calibrating and validating sizing instruments With diameters spanning Bangs Laboratories e Ask The Particle Doctor Page 10 Fluorescence Intensity Quantum FITC MESF ABC Values Antibody Quantitation DNA F P Ratio MESF Values Quantum Dots Quantum MESF Kit a range of
55. 40nm to 175um we have standards that are suitable for a broad range of sizing methodologies The standards are supplied as 1 solids aqueous suspensions in dropper bottles We also supply a wide range of diameters within our standard catalog if the higher level of traceability isn t required for every run These products include polymer 25nm 20um and silica 150nm 5um spheres supplied as 10 solids suspensions Some dry microsphere products are also available For specific sizes and ordering information see our website or catalog If you should need further clarification our friendly Customer Service Representatives are standing by well actually they re probably sitting and ready to help Catalog Code Description NTO2N NT40N NIST Traceable Size Standards size ranges from 40nm to 175pm PSO2N PSO8N Polystyrene Plain Hydrophobic Microsphere size ranges from 25nm to 20pm SS02N SS06N Silica Plain Hydrophilic Microspheres size ranges from 150nm to 5pm We require Quantum FITC calibration beads for the quantitation of FITC fluorescence intensity in MESF units It seems 3 types of Quantum FITC beads low medium high are available am confused in choosing the right one for my calibration work Can you help The different levels of kits are intended to span the intensity g CDB Lymphocytes N CD4 Lymphocytes range of common cellular analyses Low level kits are commonly iia used for cells with low expression levels or for s
56. Ask The Particle Doctor A compilation of the Questions You ve Asked over the years wo Pangs Laboratories Inc Bangs Laboratories e Ask The Particle Doctor Page 2 Alexa Fluor amp need to calibrate the fluorescence of cells labeled with Alexa Fluor 555 How should I go about doing this is it possible to use one of your Quantum MESF kits perhaps with some sort of conversion factor Quantum MESF In addition to quantitative kits for standard flow cytometry fluorochromes FITC PE PE Cy 5 etc we Quantum Simply currently manufacture Quantum MESF products for Alexa Fluor 488 and Alexa Fluor 647 Unfortunately Cellular these wouldn t be suitable for Alexa Fluor 555 as the same fluorochrome must be presented on beads and cells for quantitative determinations However if you are working with antibodies our Quantum Simply Cellular amp QSC kits may offer a solution QSC bead populations are coated with increasing amounts of capture antibody that the user then labels with the specific fluorochrome conjugated primary antibody or indirect staining may be conducted if a labeled primary mAb isn t available Bead populations are calibrated in terms of their Antibody Binding Capacity ABC or the number of primary antibodies that they will bind When the standard curve is drawn in QuickCal provided with the kits ABC assignments may be made to labeled cell populations If monovalent binding is assumed the
57. COOH beads goes into WSP they could only account for 50 of acid dose in or on the beads That is why we always recommend that you clean your microspheres before use so you don t bind to WSP and lose your precious ligand when you wash after binding If you don t want to wash before binding to save time then you will pay for that time saving with extra protein coupled to WSP and discarded Note that after removal of WSP your ligand will be firmly attached to the particle bound COOH groups Q Are all the COOH groups on the outer surface of the microsphere or also within the microspheres Surely some of the carboxyl groups are buried beneath the surface However the microspheres are non porous and therefore the titration data is representative of the carboxyl groups which are present at the outer surface and available for covalent coupling Do you know of a good system to automate small bead assays that doesn t cost a zillion dollars Any ideas You bet Try quantitative turbidimetric or nephelometric assays to measure agglutination of 100nm Ab coated beads by small samples of all your antigens Bangs Laboratories e Ask The Particle Doctor Page 39 ProActive Turbidimetric Nephelometric Assays DNA Adsorption References Silica Microspheres COOH Modified Beads Ligand Orientation Bead Characteristics Particle Determination Replicate Lots See new TechNote 304 If you have no luck with or enthusiasm f
58. ESF Molecules of Equivalent Soluble Fluorochrome units Telomere length measurements via Flow FISH To accomplish this the fluorescent bead set is run on the flow cytometer using the same instrument settings as for the labeled samples The median channel values fluorescence intensity for the beads are entered against their assigned MESF values using the QuickCal analysis template that we provide this generates a calibration curve relating fluorescence intensity to MESF value Channel values for the samples may then be entered into QuickCal to obtain their MESF assignments References 1 Baerlocher G M P M Lansdorp 2003 Telomere length measurements in leukocyte subsets by automated multicolor flow FISH Cytometry Part A 55A 1 6 2 Spiro A M Lowe 2002 Quantitation of DNA sequences in environmental PCR products by multiplexed bead based method App Environ Microbiol 68 2 1010 1013 Bangs Laboratories e Ask The Particle Doctor Page 16 Aggregation I am interested in developing an immunoassay using 100nm microspheres Could you offer me some tips for working with this size of particle Bead Handling Yes don t Just kidding although it is important to note that small polymer microspheres x 300nm Dialysis present unique challenges and there will be special handling considerations Filtration Particles in this size range are more prone to aggregation than larger spheres due to their very high surface ar
59. NA and attach these to our ProActive streptavidin coated microspheres See TechNote 302 and Kathy Turner s 2000 AACC OEM Lecture available on our website We can also talk about custom coupling your favorite material to our beads We don t offer microspheres with DNA directly attached to the surface but do have various types of microspheres to which DNA can be easily attached 1 Our Bangs Laboratories e Ask The Particle Doctor Page 34 Absorbance Q Upon agglutination of 0 8um beads the absorbance went down and I expected it to go up Agglutination Kinda like the stock market eh you didn t expect a downturn Let me reassure you that what you saw was Light Scattering probably normal You didn t say what wavelength of light you were using but there are two possibilities Assays 1 If the bead diameter is much lower than the wavelength of light used then the beads may not scatter light very well e g 100nm particles and 300nm light On agglutination however they grow to a size where they do scatter light and the absorbance will increase 2 Beads which are larger than the wavelength of light will scatter that light well e g 800nm particles and 400nm light On agglutination there are fewer scatterers around to scatter the light and the absorbance drops Thus it is normal for absorbance to drop on agglutination unless you were using far out infrared light Feel better now Particle Determination Weare interested i
60. Uniform Latex Particles p 26 27 Leigh Bangs used the diameter of the centrifuge at the top of the spinning tubes or bottles D and the height h of the liquid level in the bottles Note also that G forces are at a minimum at the top of the liquid but become higher as beads settle to the bottom of the tube since the effective diameter of rotation is larger at the bottom Thus G force increases linearly from top to bottom of the tube so the average G force is at the midpoint of the tube If you pick the top of the liquid for calculating you are safest since the beads will settle faster than that not slower See TechNote 206 for settling velocity equation incorporating G 5 59 x 105 n D where G multiples of earth gravitation constant G forces n rotation revolutions per minute rpm and D rotor diameter to top of liquid cm Dear Particle Dude we have a sequencing lab and are looking for an easy way to clean up excess dye terminators from our sequencing reactions Do you have any ideas Give our streptavidin coated beads a shot You will need to use biotin labeled primers easily made or obtained from a vendor for the sequencing reaction When the primers have extended to the terminator they can be removed from the reaction vessel by binding the strands to streptavidin coated beads and subsequently Bangs Laboratories e Ask The Particle Doctor Page 38 Custom Coupling IgG Coated Beads ProActive Streptavidin C
61. X objective to determine what isn t being spun down Also examine the tube which may have a characteristic smear on the wall if the beads are sticking to it If centrifugation isn t an ideal method or is contraindicated due to small bead size see the other separation methods that are described in our TechNote 203 Washing Microspheres I would like to purchase a fairly large batch of polystyrene microspheres but am concerned about stability What shelf life can expect A We don tassign an expiration date to uncoated polymer microspheres Polystyrene microspheres should be chemically stable indefinitely provided that they are stored under suitable conditions e g in their original buffer at 2 8 C Conditions that would be damaging to the product include freezing irreversible aggregation high temperatures gt 95 C or exposure to organic solvents swelling deformation sticking of beads In general our primary concerns for long term storage of uncoated polymer microspheres include ensuring that they do not become contaminated e ensuring that they are well dispersed before use Though many suspensions contain an antimicrobial they should be handled with care aseptic conditions where possible and stored at 2 8 C to avoid contamination proliferation of microbes If a contamination occurs it may be possible to decontaminate the suspension see PDS 726 Decontaminating Polystyrene Microspheres To ensure that spheres are
62. ability to the strept avidin molecule we know that many enzymes are more stable once immobilized 5 As mentioned before we have had clients successfully use our streptavidin coated microspheres during thermocycling We are not aware of any studies that cite use of deglycosylated avidin coated beads during PCR thermocycling Nor do we know of any studies that evaluated the activity of streptavidin avidin or modified avidin molecules after denaturation and subsequent renaturation i e studies that investigated folding errors during renaturation or the specific effects of different denaturation procedures 6 Reznik et al 1996 Streptavidins with intersubunit crosslinks have enhanced stability Nat Biotechnol 14 8 1007 1011 PubMed 9631041 7 Avidin Biotin in Pierce Catalog and Handbook Pierce Chemical Company Please note We do recommend that beads be washed prior to use as some preservatives and stabilizers can inhibit PCR need magnetic beads for my application Why should I choose yours mean what s so great about these new COMPEL beads other than the very compelling name Are you kidding Have you seen the micrograph Those babies are beautiful OK maybe it takes a real bead aficionado to truly appreciate the attractiveness of a round mostly plastic ball But seriously the COMPEL beads have several features some of which can be seen in the image which make them ideal for a whole range of appli
63. act with thiols See our TechNotes 205 Covalent Coupling and 102 Magnetic Microspheres 2 Our largest magnetic microspheres are 8um Orders may be placed through our website or by contacting our Customer Service Department 3 If you can biotinylate your protein then you could choose our streptavidin coated ProActive magnetic beads Ask if you need help As a complete novice to beads would like some advice on how to coat them with Concanavalin A Passive adsorption On what kind of beads Or is covalent coupling a better option Wow three questions this time 1 For general advice on handling beads and especially covalent coupling vs adsorption see TechNote 201 Working with Microspheres Passive adsorption on PS beads works best with larger proteins like IgG which stay adsorbed otherwise try COOH modified beads and EDAC coupling See TechNotes 204 Adsorption to Microspheres and 205 Covalent Coupling BioMag amp Plus Concanavalin A are also now available Why do the 50nm COOH modified fluorescent dyed particles clump when I try to couple protein to them Is it the dye The inability to successfully couple to the small COOH modified particles is somewhat puzzling Surely it is more difficult working with these tiny particles but you should be able to keep them dispersed in surfactant You raise several interesting points Bangs Laboratories e Ask The Particle Doctor Page 33 Binding IgG COOH Modified
64. ade by two different processes A amp B were made with surfactant and C was made with near zero surfactant Our hypothesis is that with A amp B the protein adsorbed into the proper orientation Fab parts up and Fc part down but in the third case the protein adsorbed face down or Fab buried If we are right then adding 0 1 anionic SDS or nonionic Tween 20 surfactant to C might fix the problem Conversely if you cleaned up A amp B removing the excess surfactant then they might bind the IgG face down also Q What else could I do to fix this problem You might reverse the order of addition in the binding steps Most folks doing covalent binding to CML will activate with EDAC clean to remove excess EDAC add protein to activated beads and finally wash to remove excess protein Your method was obviously successful for latexes A amp B but with C it seems notto be working Please let us know how these ideas work out and we can share the results here next time Important take home messages for all you bead binders 1 2 Not all beads are created equal They might have the same specifications of diameter standard deviation and acid content but they could still be made in many different ways We ll be glad to explain these differences to you You need to know what kinds of beads you are using and whether different bead lots are replicates or made by different producers or different processes using acrylic o
65. amber for instruments that are intended to measure single particles thereby ensuring more accurate results Once you have had an opportunity to review the user manual for your instrument and perhaps speak with Particle Technology Laboratories in Chicago our expert guide in this area please feel free to contact us for the selection of standards that are suitable for your instrument Q want to send some microspheres to a colleague in Spain for a limnology project in Antarctica Can they withstand 3 4 days without refrigeration The short answer is Yes but The longer answer is It depends And the more correct and complete answer is that to minimize any microbial growth we always recommend that beads be stored refrigerated at 2 8 C in conjunction with the use of preservatives such as sodium azide or merthiolate Room temperature storage is acceptable too If uncoated beads are stored properly and handled by aseptic techniques they should have a shelf life of more than 5 years Anyway it should be possible to ship from the US to Spain without refrigeration but you could add some ice to the package to ensure cooler travel In Antarctica s harsh climate the bigger hazard will be freeze damage to the beads Plain singly dispersed beads can be turned into badly clumped beads resembling cottage cheese if they are frozen So don t sweat the bugs down there but do guard against freezing Q What s the best way to optimize bin
66. aphics These are dot plots of the FITC PE Compensation Standard The one on the left was acquired on a BD FACScan with the compensation circuits turned off Note the circled population See how the fluorescence carry over from the FITC makes the FITC bead pull away from the axis and appear to have some PE fluorescence The dot plot on the right was acquired on the same instrument after the compensation was set F porencence I l m planning the development of an immunoassay using BioMag but I ve never worked with the particles before Where do begin You might consider working with one of our BioMag immobilization kits These include most of the things that you ll need to get started such as BioMag particles chemical crosslinkers buffers and in some instances a reaction vessel and magnetic separator You will also be supplied with a detailed protocol providing step by step instructions for coupling the biomolecule of choice to the particles and for determining coupling efficiency Once you have exhausted the supply of reagents provided with the kit you may buy the components on an individual basis Why do I have to log on to your website to access QuickCal and what is the QuickCal Access Number for Will my access number stay the same or will it change with every order By logging into our website and using the QuickCal Access Number printed on your Quantum MESF or Quantum Simply Cellular kit our c
67. articles to create a disulfide linkage By the way this disulfide bond is later cleavable with dithiothreitol DTT to release a protein or peptide if desired You can also create thiol activated particles by cleaving the disulfide bond with DTT after the first SPDP activation reaction above Hydrophilic We are trying to make a test where coated dyed particles move along a strip until they are immobilized Membranes by an antibody bound on the strip Any new ideas for drying particles on strips so they will move later Lateral Flow Tests The problem has been to pick or spot treat a membrane which will adsorb the capture antibody at the site where the colored line is supposed to form and which will release the dried particles so they can move Strip Tests down the strip to form a sandwich test One idea is to dry the particles on a hydrophilic pad which overlaps the strip upstream of the adsorbed protein When the pad is wet with sample the particles are transferred from the pad to the strip and along the strip to the antibody stripe where they are immobilized e g Fusion 5 membrane from Whatman Antibody Orientation When you use antigen coated particles for immunization do certain epitopes typically get hidden or exposed with PAPP s M Spacer Arm a different Ig response profile than a typical immunization with dem en L YY or without adjuvant followed by a boost l l Certainly if antigen is adsorbed on a surfac
68. atings to capture cells that have been antibody labeled Catalog Code Description BM595 BioMag anti Human CD2 BM580 BioMag anti Human CD3 BM581 BioMag anti Human CD4 BM583 BioMag anti Human CD8 BM596 BioMag anti Human CD11b BM584 BioMag anti Human CD14 BM585 BioMag anti Human CD16 BM586 BioMag anti Human CD19 BM587 BioMag anti Human CD34 BM588 BioMag anti Human CD45 Bangs Laboratories e Ask The Particle Doctor Page 18 BioMag COMPEL Phage Yeast Library ProMag Recombinant Antibodies References BioMag Plus Amine BioMag Plus Carboxyl Coupling Antibodies Custom Services PolyLink Protein Coupling Kit Full Spectrum Optical Alignment Quality Control Reference Beads BM589 BioMag anti Human CD56 BM590 BioMag anti Human CD71 BM592 BioMag anti Mouse CD4 BM593 BioMag anti Mouse CD8a BM594 BioMag anti Mouse CD45R BM597 BioMag Human CD3 T cell Enrichment System BM598 BioMag Human CD4 T cell Enrichment System BM599 BioMag Human CD8 T cell Enrichment System Q Do you have any products that are suitable for selecting recombinant antibodies from a large antibody phage library Magnetic microparticles have been utilized with success for efficient selection of surface displayed molecules from phage and yeast libraries Sequential magnetic bead and flow cytometric sorting has also been reported A few pertinent references follow Feldhaus M J et al 2003 Flow cytometric isola
69. ation in staining Using a fluorescent bead standard with each run can help in identifying one off sample preparation problems etc For example use of a suitable Fluorescent Reference Standard would provide a reference point for each run Getto know your instrument Quantitative fluorescence analyses probably won t be accurate or reproducible if there are problems with instrument linearity resolution etc And of course we ve seen our share of troubles over the years These are often the result of sub optimal conditions or basic errors sometimes even of the forehead smacking variety Don t worry we ve done them too And if error isn t to blame there are often simple strategies to improve results No or poor fluorescence Ensure that the primary mAb species is suitable for the kit For example the anti Mouse kit is intended to bind mouse mAbs not for the analysis of mouse cells Protect the fluorochrome conjugated Ab and stained samples from light to prevent photobleaching Ensure that the laser and detector are suitable for the reporter fluorochome In the special case of Fc tagged proteins they should be tested to ensure acceptable binding to the Fc specific antibody coated on the beads We have known some Fc tags to exhibit different binding than their native Ab counterparts and a lack of binding in rare instances Broad fluorescence peaks Use of an indirect staining approach will lead to broader peaks if this occu
70. ave 0 or 1 bead per droplet Then when the droplet dries each bead will dry as a single bead without touching another bead until they are dry This will lessen the chances of them sticking together in the dry form Contact TSI for aerosolization equipment and information Web www tsi com Fluorescent Beads What do you know about using dyed microspheres for regional blood flow tracing Regional Blood Flow Tracing We have visibly dyed and fluorescent microspheres of all sizes 20nm 20um and we are continually making more at customers requests Undoubtedly these may be used for many applications Look for Dyed and Fluorescent Microspheres pages in the Products amp Ordering section of our website www bangslabs com Bangs Laboratories e Ask The Particle Doctor Page 45 We don t have experience using our microspheres in regional blood flow experiments Please note that our microspheres are not supplied as sterile suspensions The real experts in that area are the people at the Fluorescent Microsphere Resource Center at the University of Washington They have developed a technical manual describing fluorescent microsphere technology for regional blood flow applications Their Web home page is lt http fmrc pulmcc washington edu gt Dr Robb Glenny will be able to help you Email glenny u washington edu Adsorption Binding Issues Filter Screening Membrane Challenge Small Microspheres Hemagglutination Tests Reverse
71. beads e Quantum MESF kits are not limited to antibody based systems For example they have been used for DNA based applications such as telomere length determination e The MESF unit is a standard fluorescence intensity unit However if you wish to report the number of antibodies bound the effective Fluorophore Protein ratio effective F P ratio or labeling density of the antibody conjugate would need to be determined This may be accomplished by staining our single population Simply Cellular product calibrated in terms of ABC with the antibody conjugate and determining its MESF value by running it against the appropriate MESF kit The MESF value is then divided by the ABC value to obtain the effective F P ratio of the conjugate Quantum Simply Cellular e Kits are available in anti Mouse anti Rat and anti Human versions for use with mouse rat and human monoclonals respectively e Beads are calibrated in terms of ABC For cellular analyses if you presume monovalent binding of antibody to the cell surface receptor then ABC antibodies bound marker density This circumvents the need to determine the effective Fluorophore Protein ratio of the conjugate e Antibody conjugates with any type of fluorescent label may be used including less standard fluorochromes and quantum dots e Beads are labeled by the user with the same antibodies that are used to stain cells This presents the benefit of having the identical conjugate on b
72. cations The first observation to make is that the beads are spherical There are no nooks crannies or crevices in which to waste costly antibody oligonucleotide or other ligand where it might be inaccessible and unable to do its job Next you will notice that COMPEL beads are not perfectly smooth The slight surface roughness effectively increases the surface area allowing you to bind more ligand to the surface than you could to a smooth sphere Because of this property the COOH group surface titer and protein binding capacity are much higher than expected for a smooth bead In our hands 3um diameter COMPEL beads bind slightly more streptavidin than the same weight of um diameter magnetic beads Finally you can note that the beads are all the same size All sizes of COMPEL have extremely uniform size distributions This property makes separation predictable and easy You won t have to wait for the small beads from a heterogeneous population to straggle over to the magnet Additionally COMPEL beads reflect a nice tight population on our flow cytometer What you can t see in the image is that COMPEL beads respond very quickly to a magnet and redisperse readily when the magnet is removed And although they move to the magnet rapidly their physical density is low enough that they remain suspended in solution long enough for your binding reactions to take place What a great package of features for little mostly plastic balls We encou
73. cence These are used to set up a standard curve relating fluorescence intensity to Antibody Binding Capacity Once you ve generated that curve you can read directly from it the ABC values of all the samples that you run Just like the Quantum Simply Cellular anti Mouse Antibody Binding Standards the new product is offered in 20 100 and 280 test sizes Don t let the anti human name fool you the beads are actually quite friendly Q How do you avoid freezing especially during the winter months I dress warmly myself And we dress our microspheres in packaging and proper labeling to protect them from freezing which is effective except where they are inadvertently left out on a loading dock over the weekend or advertently intentionally put in the freezer by some helpful receiving department person to keep them safe overnight We also carefully choose the shipping method and timing shipping by overnight carriers who will handle our products carefully and get them to you before they can freeze In Freezin Season we do not ship on Fridays so your package will not sit on a loading dock over the weekend anywhere You will have to worry about that guy in receiving though Help My microsphere reagent has lost activity only one month after adsorbing IgG and blocking BSA What happened What can do A Your problem with reagent stability could be due to one or more of the following 1 Concentration of BSA in storage buffer
74. cence Intensity esses 10 22 Fluorescence Quantitation sssseesese 14 Fluorescent Beads sussss 5 6 15 16 45 60 Fluorescent Reference Standards s 12 14 aA Terme oesau 3 Freezing Prevention sese 25 29 Full Sp Ctrum innne 18 Functionalized MicroSpheres ccccccsecsseesseeessseesseeeeeees 20 G G FOICBS end eee et idee RAP e retta 37 53 GFP duci E 26 GlacialiBlie eie hec e tee erc 4 16 Glass Transition Temperature seeeeee 37 Gravimetric Analysis sse 36 H Handling Beads senes 16 Hemagglutination Tests sese 45 High Throughput Format enne 11 Hoen 12 Hydrophilicity oeerrrrverrvrrrrrororererrrvorereresreverensesrsvonesennnne 22 Hydrophilic Beads 40 61 Hydrophilic Membranes eene 54 59 Hydrophilic vs Hydrophobic Surfaces 64 Hydropho bit iy eese 5 l IgG Coated Beads 38 fenta Mno asl nl Soh ie Goes rrt cea 29 IgG Coating Levels ssseeennnnnen 25 IgG Spacing sss 35 IMMUNOASSAYS ccecescsccsessccsececssessececsecsseseesecsecsueaeenes 21 60 Immunophenotyping ener 19 mad 12 Initial Target Channels soeoovorerornorororesvsrsvoresrrrsverer
75. ch you would probably need to purify the serum to increase the efficiency of the biotinylation step Another approach would be to use our uncoated COOH functionalized microspheres By first purifying the serum you could attach the antibodies of interest to the microspheres via covalent coupling reaction see TechNote 205 For protocols to work with streptavidin coated microspheres see our TechNote 101 Download from our website We can also talk about custom preparation of GAR coated beads Q We have some of your surfactant free carboxyl modified particles P S V COOH Are the COOH groups strongly bound to the particles or may some of them be released The COOH groups are a result of a copolymerization of styrene and a vinyl carboxylic acid such as acrylic or methacrylic acid In most cases the microspheres are sold diluted with deionized water to 10 solids Thus surfactant if any buffer salts for pH control counterions and especially water soluble polymer WSP remains in the soup No surfactant was used in your beads but there are other solutes WSP is probably composed of styrene acrylic acid AA copolymer molecules which have enough AA to make the polymer chain soluble enough to leave the particle If methacrylic acid is used the WSP will be significantly reduced due to the lower solubility of MAA and its copolymers with styrene About 15 20 years ago some Dow guys found that about half of the AA added to make
76. cles via biotin streptavidin really want to do this with non magnetic beads since they are easier to keep in suspension during the reaction Will non magnetic beads handle the extreme temperature cycling IF PCR works with our mag beads then you should have no problem with our other microspheres The magnetic microspheres are made of polystyrene PS or styrene divinylbenzene S DVB copolymers with 6 90 DVB The glass transition temperature GTT of pure PS is 105 C well above the normal PCR maximum temperature of 95 C So any of our magnetic or non magnetic PS beads should survive PCR Since GTT increases with DVB content ask for a chart of DVB content vs GTT if you have any doubts about or problems with the PS beads then you can also choose plain non magnetic microspheres with various DVB contents How many G s should I use to centrifuge 350nm microspheres Someone recommended 3200rpm for 20 minutes That s about 2000 2400G In order to really specify how to centrifuge microspheres one must describe the centrifuge geometry and speed n rpm If someone says only Centrifuge at 3200 rpm you really don t know what to do since you don t know the diameter of the rotor or the distance the beads must settle Likewise if they say Centrifuge at 2400 G s you still don t know how far the beads are to settle When you know the geometry then you can decide how long you should spin the beads In his book
77. compare to the work I ve already done I m worried about the difference in F P ratios between the two antibodies Fluorescence Intensity While the F P ratio fluorochrome protein ratio the number of fluorochromes Quantum Simply per antibody will differ between the two antibodies that difference will not Cellular be reflected in the Antibody Binding Capacity ABC results obtained from the QSC kit This is also due to the fact that each of the beads in the Quantum Simply Volume 16 2 Cellular kit is calibrated to bind a specific number of monoclonal antibodies June 2003 regardless of the F P ratio of the antibody You may notice a difference in the fluorescence intensity of both your samples and the QSC beads with the new ERE es antibody but as long as your samples and the QSC beads are stained with the same o 97 uniform silica beads antibody the results should correlate very well with your previous work Adsorption Q I am familiar with the use of plain polystyrene for adsorption of protein specifically antibody I have always heard that silica does not bind protein If this is the case what is its use Hydrophilic Silica has become the substrate of choice for many applications due to its low nonspecific protein Nonspecific Binding binding properties It is hydrophilic and is less likely to adsorb high amounts of protein nonspecifically than hydrophobic polymer such as polystyrene Additionally its negative charge will
78. considered here At what percentage solids did you perform the coating Working with particles at 0 5 1 solids can help reduce the chance for clumping If your particles were coated after they became clumped then you may have great difficulty in trying to break up the clumps The only possible solution that we ve come up with is to add a large amount of your protein to the coated microsphere suspension After adding the extra protein sonicate the suspension to try to break up the clumps If the clumps do break up the extra protein should coat the single particles before they have a chance to stick back together Q How can I get your particles to get down in our centrifuge A It sounds as if you tried but failed Please keep the following points in mind 1 99 99 of our products are heavier than and will settle in water see density column in our product list The maximum settling velocity of a single sphere settling in water cm sec v 5 448 x 10 p 1 d where p density of the solid sphere g cm and d particle diameter um Where many particles are settling together they interfere with one another and at 5 solids the hindered settling velocity v 2 3 v Thus polystyrene particles p 1 05 g mL settle in water but will not settle in solutions with densities gt 1 05 g mL In a strong Bangs Laboratories e Ask The Particle Doctor Page 53 Settling Rates Washing Beads Diagnostic Test Desi
79. coupling and then there is no large blocker protein which might cover the small molecule binding site What is the best way to bind antibodies to microspheres What about adsorption covalent coupling secondary antibody binding and Protein A 1 Directadsorption is the oldestand simplest method for putting proteins onto polystyrene particles It works well and most proteins will stay on the particles forever or at least for a long time See our TechNote 204 for protocols to start you off 2 Many people prefer the more secure covalent coupling of proteins Water soluble carbodiimide binding to carboxylate modified particles is a well established method for covalent coupling See TechNote 205 for protocols and other covalent binding options 3 Ifyou adsorb one Ab and use it to bind another Ab e g goat anti mouse secondary and mouse monoclonal primary the second Ab will certainly be more accessible sticking further out into the aqueous phase It may also be oriented more favorably and therefore significantly more active 4 You can also use proteins A or G to attach Abs to particles Some claim superior orientation this way Since protein A binds specifically to the Fc portion ofthe IgG the Fab portions of the Ab are pointed away from the surface You have a choice of native proteins A or G recombinant forms of proteins A or G these have deleted sequences for reduced nonspecific binding potential or even with recombinant fusion pr
80. d beads should be washed out of it before coating to avoid interference with binding Washes may be performed in a generic buffer such as PBS or whatever buffer will be used for the next step in the coating process such as our Bangs Bead Coupling Buffers BUFF1 BUFF4 As with any wash steps if these which effectively reduce the concentration of stabilizers lead to aggregation a small amount of stabilizer may be added back in to treat stickiness The suspension may also be vortexed or carefully sonicated to disrupt aggregates For more on washing in general download TechNote 203 Washing Microspheres from the Technical Literature portion of our website Q As polystyrene is a hydrophobic material how are polystyrene microspheres stabilized A Polystyrene microspheres in general are stabilized through endogenous charge groups and surfactants Charge Groups PS beads are often synthesized with use of a charged e g sulfate based initiator which will impart residual groups e g S04 on the surfaces of beads Beads that have been specifically functionalized as PS COOH which will have a carboxyl monomer co polymerized with styrene will also have that charge group on the surface Surfactants PS beads are commonly synthesized in the presence of surfactant which is essentially a wetting agent Surfactants are polar molecules with hydrophobic and hydrophilic ends The hydrophobic tail associates with the hydrophobic bead surface the hydro
81. d low background in these regions of the spectrum As a final note another strategy would be to use off peak excitation which will reduce fluorescence output including the carryover signal Fluorescence carryover is the region of overlap of emission spectra I just purchased Quantum Simply Cellular microspheres for the first time and want to be sure that I m using the correct amount of antibody for labeling what is your recommendation As Quantum Simply Cellular amp microspheres are intended for quantitative analyses it is imperative that the beads are stained to saturation We recommend that an antibody titration be performed for the beads to determine optimal antibody concentration bear in mind that the amount of antibody needed to saturate the beads may be different than the amount that is appropriate for the cells If the antibody concentration is not reported for the conjugate I would suggest contacting the supplier They should be able to provide this value which will aid in establishing the range of concentrations to be used for the titration Keep in mind however that the reported antibody concentration may include the fluorochrome and a weight based concentration of PE conjugated antibody MW of PE 280 000 Da will mean far fewer antibodies on a number basis than the same amount of FITC conjugated antibody MW of FITC 389 Da need compensation beads for flow cytometry and currently use your Simply
82. ded ask the chemist if he she has or can make any chromosulfuric acid This is made by mixing potassium or sodium dichromate into concentrated sulfuric acid Certain chemists have used this reagent to clean glassware and it chews up anything organic It will etch and oxidize the bead surfaces making them hydrophilic It will probably make them disappear if they are left in there long enough It is possible to treat the beads with an electrical plasma in the presence of gasses like 0 NH etc to create hydrophilic surfaces but have no experience with this Some of our beads are made by suspension polymerization processes which put an inorganic coating on the beads These will have natural hydrophilicity Apparently your beads were not made this way Q With all the concern about health now I just want to be sure Are your particles grown organically We assure you that our polystyrene microspheres are 100 organic and our silica beads are 100 inorganic Q Regarding chloromethyl latex do you have any comments about good storage conditions for this bead after protein coupling As far as we know chloromethyl latex should be very stable after binding First the chloromethyl groups are reacted to form a stable covalent bond with protein Second the protein coating should impart colloidal stability Main Question Will Tween 20 be sufficient to prevent protein adsorption to the derivatized bead or should I use a neutral pro
83. ding of biotinylated oligos to your streptavidin coated beads while minimizing NSB A customer recently advised us about his use of a bicarbonate buffer for the binding of biotinylated oligonucleotides to our SA coated microspheres which in his hands effectively eliminated NSB nonspecific binding For buffer and binding conditions see Bianco P R L R Brewer et al 2001 Processive translocation and DNA unwinding by individual RecBCD enzyme molecules Mature 409 374 378 Bangs Laboratories e Ask The Particle Doctor Page 30 Streptavidin Coated Microspheres Blockers PEG Protein Adsorption References Loss on Drying Solids Concentration Turbidity Washing Beads Other conditions that have been said to reduce nonspecific binding include increased salt concentration increased pH decreased probe concentration or decreased streptavidin binding on the bead Please note that we do not routinely work with biotinylated oligonucleotides in house and thus have not optimized binding conditions for such Q How do attach PEG to microspheres to deter prevent adsorption of cytoplasmic extracts or other protein adsorption There appear to be several articles in the literature about attaching PEG via covalent coupling or adsorption to inhibit protein adsorption Here are two articles that might be of particular interest Satulovsky J M A Cargnano I Szleifer 2000 Kinetic and thermodynamic control of protein ad
84. ds they would be very easy to spin down and to resuspend Larger beads will also settle easily and rather quickly without centrifuging the larger the bead the faster they will settle of course They will also settle faster in isopropanol than in water You can do dead end or bed filtration of large beads more easily too Flow through a filter cake is much easier with 1um beads than with 0 1um beads But since they have a lower specific surface area you might need to use more beads would suggest choosing the size by what surface area you need and by how easy it will be to clean them before and after coating For more information see our TechNotes especially 201 203 and 206 How do I bind a protein via the protein s carboxyl or amino terminus to 2 9um magnetic beads and then use these beads for pulling out antibodies that bind to protein I don t want to use a chemistry that might modify the cysteine residues on my protein Also would it be easier to use preactivated beads that can just react with my protein so I don t have to do the chemical reactions myself You folks keep sneaking in two questions OK this time 1 To avoid the possibility of binding to the cysteine residues on the protein try our COOH functionalized mag beads Activate the beads with EDAC and NHS wash the microspheres and add protein Unlike the o acylisourea intermediate formed by the EDAC reaction the succinimidyl ester formed by the NHS will not re
85. du hmarchiv cytomail htm Email archives are also available for searches Q We are a contract lab that receives many requests for magnetic particle based DNA isolation services particularly for genomic DNA from whole blood We need a product that will provide exceptional yield and is amenable for use on our high throughput automated platform Can you help Our SNARe Whole Blood Genomic DNA Purification System Catalog Code BP691 features our patent pending DNA Separation Particles for the efficient isolation of dSDNA We offer protocols for both manual microcentrifuge tube and automated 96 well plate formats so that you may scale up with ease Both protocols result in exceptional yield 200g DNA 200uL tube whole blood or 5 20ug DNA per well 100uL lysate from fresh or frozen whole blood WBCs or MNCs Protocols are provided in Product Data Sheet 691 microcentrifuge tubes and Product Data Sheet 691A 96 well plate We also offertwo other DNA purification systems see below as well as variety of rare earth magnetic separators forusebeforescale uptoahighthroughputsystem Ourseparators are designed to accommodate acomplete range of magnetic separation applications including cell sorting mRNA and DNA isolation and purification of biomolecules Catalog Code Description BP691 SNARe Whole Blood Genomic DNA Purification System BP692 SNARe Plasmid DNA Purification System BP693 SNARe Plant Genomic DNA Purification System LS001
86. e PCR Thermocycling Centrifugation G Forces Settling Velocity DNA Sequencing Dye Terminator Reaction Purification Streptavidin Coated Microspheres A My guess is that the boiling step would denature the protein antibody immunoprecipitate In any case it would likely soften the polymeric microspheres used to separate the complex Beware of irreversible clumping of soft microspheres I think that you would have better luck attaching biotin to the chicken antibody and attaching these to our streptavidin coated microspheres While the streptavidin biotin bond is strong it can be broken more easily than a covalent attachment See Biotechniques 26 249 254 1999 for a discussion on a procedure for using 2 mercaptoethanol to disrupt this bond Another option might be to attach NHS Iminobiotin from Pierce rather than biotin to the chicken antibody Iminobiotin has a lower affinity for streptavidin and therefore can be displaced simply by controlling the pH The folks at Pierce can probably supply an elution protocol Our TechNote 101 download directly from our website www bangslabs com lists a protocol for attaching biotinylated ligands to our streptavidin coated microspheres We have other ideas such as binding the biotin to the Ab s using cleavable linkers then binding Ab s to SA beads Please ask for more information if you are interested Q have gotten PCR to work with one of the primers conjugated to your magnetic parti
87. e the epitopes on the down side will be less likely to stimulate Ig response You might want to covalently bind molecules so the desired epitope s are face up Alternatively you might want to put the antigen on the end of a tether or spacer arm like amino caproic acid Agglutination Assays Q Can you make a sandwich strip test for small molecules with dyed particles Dyed Microspheres A Any molecule with MW lt 6000 are too small to allow binding by two antibodies simultaneously thus all assays on mycotoxins and other small molecules such as steroid hormones use competitive assays Special Guest Response by Dr Roger Collin Mycotoxin Research Group AgResearch New Zealand Sandwich Strip Test Dry Silica Q Why do you ship some silica particles dry and how do I redisperse them Redispersing Beads We grow silica particles from an alcohol solution Because it is difficult and expensive to ship them in that Silica Microspheres flammable solution we usually evaporate the solvent and ship them dry or resuspend them in deionized water for shipment To disperse them in water we recommend breaking up the dry material by grinding with water in a mortar and pestle plus use of an ultrasonic probe to assist in thorough redispersion Don t worry you can t break the individual particles Lateral Flow Test Q How can I make a direct strip test for small molecules Sandwich Strip Test A If you can raise Ab s to two different epitopes o
88. e caught then fluorescence on the filter will be a measure of antigen content of an agglutinating sample See TechNote 103 Fluorescent Dyed Microspheres for a list of different combinations of sizes surface chemistries and dyes Dye names and excitation and emission wavelengths are listed Should we use an electromagnet or a permanent magnet to separate the magnetic particles from suspending liquid Either should work The combination of liquid path length tube diameter and magnet strength should be arranged to cause separation in a reasonable amount of time We usually recommend rare earth magnets because they are so strong An electromagnet should work as well providing that there is no residual magnetism in the electromagnet when it is turned off See TechNote 102 Magnetic Microspheres Q How do I couple protein to particles while avoiding nonspecific adsorption A Two routes have been attempted in the past 1 Adsorb or covalently couple the appropriate protein usually IgG onto polystyrene Then adsorb or couple a blocker like BSA to fill any empty surface with inert protein leaving no space for nonspecific adsorption by anything else in the sample Currently we recommend this route and can share more details of this approach Coadsorption or co coupling of the blocker protein is another option as well 2 Start with hydrophilic particles with surface functional groups but which have very poor adsorption capacity
89. ea volume ratios and may require more surfactant and sonication than their larger diameter Small Microspheres counterparts In fact you may find it useful to sonicate the suspension before during e g every 15 minutes and after coating An automated particle sizer can aid in determining the level of monodispersity Washing Beads i e fluctuation in mean diameter as can traditional microscopy Although you will not be able to visualize individual particles with a standard microscope aggregates should be visible using 400X magnification Regarding washes centrifugation will not be a suitable means of separation as pelleting is likely to cause irreversible aggregation Rather washes are typically performed using filtration or dialysis Filter companies can provide suggestions regarding a suitable filter or dialysis membrane or cartridge i e a pore size or MWCO Molecular Weight Cut Off that will retain particles while allowing the removal of unbound antibody and blocking molecule Quantum Simply Q I routinely use your Low Level Quantum Simply Cellular amp Goat anti Mouse kits Catalog Code 814 as Cellular a quantitative tool in my lab but see that 814 is no longer available on your website HELP Don t be alarmed After our new Flow Team put the Quantum Simply Cellular QSC kits under scrutiny in support of our ongoing goal to offer the highest quality products a few changes were made 1 The Low Level 814 kit and t
90. eads and cells Please note that it is advisable to titrate the antibody conjugate to ensure that the beads are stained at saturation and it is imperative that standardized staining protocols are used to ensure consistency of results Catalog Code Description 824 Quantum FITC low level 824p Quantum FITC low level premixed 825 Quantum FITC high level 825p Quantum FITC high level premixed 826 Quantum FITC medium level 826p Quantum FITC medium level premixed 827 Quantum R PE 828 Quantum PE Cy 5 823 Quantum APC 815 Quantum Simply Cellular anti Mouse IgG 816 Quantum Simply Cellular anti Human IgG 817 Quantum Simply Cellular anti Rat IgG We are developing an automated magnetic chemiluminescent immunoassay but have had difficulty with uniform separation of the spheres Do you have any products that are suitable for a higher throughput format Bangs Laboratories e Ask The Particle Doctor Page 12 Uniform Separation A There are many factors that impact the separation of superparamagnetic microspheres including bead uniformity the form and size of iron oxide inclusions and homogeneity of magnetite distribution ProMag our new line of uniform superparamagnetic microspheres have been carefully designed and rigorously tested to ensure rapid uniform apsorbanee separations as reflected in the graph wavelength 640nm Magnetic Separation Rates Time seconds Biotinylated Olig
91. ector and the incident light beam Particle counters or sizers like those made by Brookhaven Instruments Coulter and Malvern can be used to monitor the size distribution of a population of single double triple etc particles Electrophoretic mobility instruments can be used for particle characterization and may help in aggregation studies Field flow fractionation is a technique which can sort out the relative ratios of singles twins triplets etc FFFractionation Inc of Salt Lake City UT offers these instruments now available through Postnova Analytics www postnova com See paper by Giddings J C et al 1989 Colloid characterization by sedimentation field flow fractionation VII Colloid aggregates J Colloid amp Interface Sci 132 2 554 565 In this work they measured singles twins triplets etc of latex particles which gave them to test Q What size particles should use for a latex agglutination test kit sensitivity should increase with particle diameter so we d say try the larger ones first Because they are popular sizes there is a good selection of particles in either range At 0 7 0 9um you can choose from several replicate lots of PS particles COOH modified particles with different levels of surface COOH groups are also available for covalent linkage The short answer is 0 8um The long answer People use anything from 0 1 dum The most popular sizes are either 0 2 0 3um or 0 8um In this range
92. efore any samples are run The Window positioning is accomplished by adjusting the PMT voltage with the compensation turned off such that the QC3 standards fall in predetermined Target Channels After setting the Target Channels the compensation may then be accurately adjusted using labeled control cells as they represent the most accurate spectra of the sample cells Re running the QC3 standards at these settings post compensation provides the Instrument Specific Target Channels which should be achieved daily if the instrument settings are not changed The instrument noise may then be qualitatively assessed with the Certified Blank microbead standard by comparing its position relative to the autofluorescence of non labeled cells to ensure that noise does not interfere with the assay Catalog Code Description 845 QC Windows FITC PE 846 QC Windows FITC PE PE TR 847 QC Windows FITC PE PE Cy 5 848 QC Windows FITC PE Cy 5 APC Q I conduct many different types of cell separations Do you offer any products that support this application Funny you should ask We were just talking about the variety of BioMag kits and particles for human and mouse cell separations that we offer Well as far as the boss knows that s what we were talking about A list of our anti leukocyte BioMag particles is provided below We also offer BioMag T cell enrichment systems and a range of secondary antibody and other affinity co
93. envsnener 17 LON EXCDO DOO aars e ter enacts 55 Isoelectric Points sss 61 ESIOGnu ns 32 L Large Microspheres sese Lateral Flow Test Membranes 62 Lateral Flow Tests 43 51 idu EN 54 55 58 63 Latex Agglutination 35 Latex Agglutination Tests 28 35 64 Latex Immunoassay 63 Lectin Coating 21 Lectin Coupling 36 Ligand Bead Calculation 26 Ligand Orientation 36 39 Light Scattering ASSAYS ssssssseeees 34 57 Lipopolysaccharides essen 5 Long term Storage ssssssssseseeeennen 9 LOSS ON Drying iier rne irren tectis 30 55 Low Particle Concentration seen 55 M Magnetic Beads sss 13 32 Magnetic Microspheres seeenn 2 Magnetic Particles sse 60 Magnetic Separators ssssssseennee 13 Magnetization sss 14 Magnet Selections essen 60 Maximum Adsorption Rate IgG sssss 33 Membrane Challenge essen 45 Membrane Pre Coating seen 62 Membrane Sticking essen 59 MESF Values eese 10 23 28 Microbial Growth
94. for proteins The idea is to covalently link only active protein to these particles Subsequent nonspecific binding is theoretically prevented by this method Bangs Laboratories e Ask The Particle Doctor Page 61 Particle Determination Adsorption Co Adsorption Isoelectric Points Monoclonal Antibodies Polyclonal Antibodies References Anti Microbial Contamination We continue to search for particles which are appropriate for this second approach The difficulty is to obtain a surface which does not adsorb anything but which has groups suitable for covalent attachment Possible candidates for this approach are amide modified particles with their very hydrophilic surfaces They can be partially derivatized with hydrazine to form some hydrazide groups which permit easy covalent coupling Q What size particles should use for a particle based test The diameter you choose is dictated by the type of format you want to use We suggest the following guidelines Test Assay Type Particle Size Slide agglutination 0 2 0 9um Latex immunoassay 0 01 0 3um Particle capture ELISA 0 3 0 9um Chromatographic strip test dyed particles moving along strip like pregnancy tests 0 1 0 4um depending on particle capture method gepending on porosity of the strip Q Why can t we get our monoclonal antibodies to stick onto particles as well as the polyclonal antibodies do Monoclonal Mc and polyclonal Pc antibodie
95. g APC Cy 7 We just purchased a customized cytometer and need additional bead standards for the UV and violet lasers We would also like something for the red laser with far red detection Can you direct us to suitable DAPI products Far Out Red Living life on the edge of the visible spectrum eh No problem we re edgy we can help Our line of Fluorescence Reference Standards includes anumber of products for UV excitation such as DAPI Hoechst Fluorescent Reference Indo 1 etc We also recently expanded our offerings to include specific products for violet lasers Glacial Blue Standards and for excitation with red lasers for far red emission APC Cy 7 and Far Out Red Hoechst Glacial Blue may be excited using a violet 405nm laser with detection in the blue region of the spectrum Our new surface labeled APC Cy 7 standard is our first Indo 1 dedicated standard for red excitation with far red emission See also Far Out Red introduced in July 2008 Red Laser Far Red Detection Single color Fluorescence Reference Standards may be used to QC a specific path of the optical system laser filter PMT to optimize filter and mirror sets for UV amp Violet Excitation fluorophores and to establish a test specific Target Channel Value for instrument set up Bangs Laboratories e Ask The Particle Doctor Page 13 Flow Cytometry Supplement Quality Assurance Standardization DNA Isolation DNA Purification
96. g for further discussion sample protocols The TechNotes may be downloaded from our website www bangslabs com Briefly covalent coupling of the Ab to COOH functionalized beads should result in a very stable reagent however covalent coupling protocols generally do require some optimization protein and activator concentrations incubation times temperatures buffer pH blocking Conjugation of ligand to protein coated beads generally requires little optimization if any however the initial cost of microspheres will be slightly higher and the reagent may not be as stable although this is highly dependent upon the protein coating and the bead environment The streptavidin biotin system is very stable however biotinylation of the antibody with potentially some optimization of this reaction or purchase of biotinylated Ab would be required If your Ab is IgG a covalent coupling protocol should be relatively straightforward Bangs Laboratories e Ask The Particle Doctor Page 29 Binding Capacity Streptavidin Coated Microspheres Particle Sizing Freezing Prevention Microbial Growth Shipping Methods Sodium Azide Storage Bicarbonate Buffer Biotinylated Oligonucleotides Nonspecific Binding References and published protocols are readily available If you are binding a different Ig e g IgM the chemistry will be a bit more involved Q Is it possible to bind just one biotinylated fluorophore molecule to a stre
97. gn Technical Services Cleavable Crosslinkers Disulfide Bonds Protein Binding References Thiol Activated Particles enough solution of salt or sucrose they will never settle Particles with d lt 0 6um will never settle at normal gravity because Brownian motion keeps them dispersed Any particles which are more dense than water will settle in water given enough centrifugal force The usual problem is that people don t spin fast enough or long enough The minimum G forces generated in your centrifuge can be calculated as follows G 5 59 x 105n D where G G forces multiples of earth s gravitational constant n rotation revolutions min rpm and D rotor diameter to top of the liquid cm Maximum G forces would be calculated from the rotor diameter to the bottom of the liquid in your tube while it is spinning Thus if your centrifuge has D 10cm and spins at 10 000 rpm it will generate G 5 59 x 105 x 10 000 x 10 5590 G s and your particles will therefore settle 5590 times as fast as at 1G Settling time t h v where h distance the particles must settle top of the liquid to top of the settled layer particle After they are spun down the particles may be very difficult to redisperse as single particles and you may not know this unless you are careful to monitor particle size after the redispersion step Carboxylate modified particles other charged or hydrophilic particles and protein coated particles will
98. hand silica has a very hydrophilic surface with many negative charges no surfactant to remove Therefore we would not expect protein to adsorb as well unless you can arrange for many positive charges on your protein molecules Even then adsorption will probably be reversible by changing the ionic environment ionic strength and pH Good News 1 You should be able to adsorb protein onto silica when silica is negatively charged when pH gt 3 7 and IgG is positively charged pH lt 8 but bonding is still not as permanent as on PS 2 DNA RNA will adsorb onto silica in the presence of chaotropic agents See TechNote 302 3 We derivatized some of the silica microspheres with silane coupling agents and created amino modified and COOH modified surfaces Ask for your favorite flavor Now you can covalently couple ligands onto the silica microspheres by various chemical schemes See TechNote 205 Covalent Coupling We are having trouble applying a consistent antibody coating directly onto nitrocellulose membrane Would it be better to coat microspheres and put them on the membrane That is a great idea It may be difficult to get an even coating on your membrane since the protein solution may want to wet into the membrane depth and spread out on the surface width You have no control of the adsorption reaction About 10 years ago in the early days of particle capture ELISA tests those blue dot tests like Hybritech s ICON pregnanc
99. hat incorporates a spacer such as some of the long chain tethers that are available just purchased some of your ViaCheck Viability Standards and Concentration Controls for use with my Vi CELL and CEDEX analyzers see the reported values for viability and concentration on the products Certificates of Analysis however I m not sure what I should be using as pass fail criteria when I run them on my instruments Certificates of Analysis for ViaCheck products provide formal lot specific values for concentration and viability We don t offer firm pass fail criteria for customer runs as these need to be established by each facility taking historical instrument performance and study objectives into consideration Users will often base their pass fail criteria around the formal result that we report for example 10 of our reported viability value We would encourage you to use several runs over time and on multiple instruments if you have them to establish your specifications This will allow you to get to know your instruments their capabilities and any quirks and to set meaningful specifications Q A number of our fluorescent beads seem to emit throughout the spectrum including orange and red regions even though their primary emission is supposed to be in the green Why is that and what can we do about it This is carryover It s what s known as too much of a good thing and all is lost Kidding Bangs Lab
100. have decades of shelf life really Q How is shelf life affected by incorrect storage pH and the occurrence of bacterial contamination A If pH is too low beads will have low surface charge reduced colloidal stability and consequently shorter shelf life Bugs can eat the surfactant and cause colloidal instability We or you can add antimicrobial sodium azide if you like How do you choose the different carboxylic acid groups for the bead coating you have three available The first COOH modified particles were made by copolymerizing styrene and acrylic aid We have the widest selection of these Methacrylic acid and several other monomeric acids may also be used Styrene and maleic anhydride copolymers are an alternate way to make COOH beads Maleic anhydride undoubtedly hydrolyzes to dicarboxylic acid groups These particles have been very popular our biggest seller in dollars and kilograms so we know they work well Perhaps the adjacent COOH groups have special properties We have a few particles with spacer arms of 2 or 6 carbons These were made as examples of what can be done to improve binding to particles We encourage people to try the tether concept especially with very small ligands like haptens Pierce and others sell activated spacers for use with COOH particles Bangs Laboratories e Ask The Particle Doctor Page 48 COOH Modified Beads Titration Data Coulombs per Particle Derivation Equat
101. he Medium Level 815 kit were combined to satisfy the requirements of both users and 815 simply won as the catalog code to maintain 2 The new QSC Goat anti Mouse kits Catalog Code 815 as well as the QSC Goat anti Human kits Catalog Code 816 are now being shipped in five separate bottles a blank and four different Antibody Binding Capacity ABC levels Instead of receiving your normal 1mL 5mL or 14mL premixed quantities you will be receiving 1mL 5mL or 14mL of each bead component A much better value for you Fluorescent Beads Q Do you have any fluorescent beads that are suitable for UV amp Violet excitation Glacial Blue In addition to Plum Purple we are pleased to introduce our new violet and UV excitable fluorophores Ultra Violet Dye Glacial Blue and Ultra Violet We also have a few offerings on our inventory to support applications in flow cytometry UV amp Violet Excitation Glacial Blue 360 450 Ultra Violet 360 390 INTENSITY INTENSITY 250 350 450 550 250 350 450 WAVELENGTH nm WAVELENGTH nm Our TechNotes 103 and 103A which may be downloaded from the Technical Literature section of our website provide spectra for our other fluorophores If we don t have a product on inventory that is ideal for your application we would be pleased to provide you with a quotation for a custom project Bangs Laboratories e Ask The Particle Doctor Page 17 Common Window of Analysis Daily QC Flow C
102. he relatively low MW surfactant and protein will eventually displace the surfactant It may work unless and until you change the conditions If you establish a protocol with this technique and then switch to another type or lot of microspheres you might get very different results This is because the surfactant type and level may be different If you switch suppliers you could easily get a different surfactant and or concentration and they may not tell you what or how much they used Then both the equilibrium and kinetics of your protein adsorption process may change This technique can be made to work by telling us that you need to get the same lot or recipe for your next shipment Better still would be to test the ruggedness of your procedure by trying several lots of the same microspheres while you are developing your protocols 2 Clean the particles with a dilute surfactant solution of your own choosing then coat with protein under reproducible conditions Use only enough surfactant to ensure colloidal stability but not so much that it interferes with protein adsorption Remove sweater in a warm and comfortable environment 3 Clean microspheres in the presence of protein This is a much more active version of option 1 above Add protein to raw microsphere suspension and dialyse or ultrafilter the surfactant out We first heard of using low maybe 10 000 or 50 000 MW dialysis tubing which would let our surfactant but retain the protein A
103. he self assembly of lipid bilayers e As spacers for flat panel displays e Select lots of highly uniform silica have also been used for microstructure assembly Flow Cytometer We have flow cytometers in labs at several different facilities Is there a way to correlate the values of samples between the different instruments Quantum MESF Yes You can use Quantum MESF standards to establish standard curves relating channel values Standardization to fluorescence intensity on each instrument By determining the intensity of your unknowns in MESF Molecules of Equivalent Soluble Fluorochrome units you will be able to compare the results of samples run on different machines Antibody Quantitation Q How do MESF units relate to the number of antibodies binding to a cell F P Ratio A There is a direct relationship between the MESF value of a cell and the number of antibodies bound to it The MESF value however does not equal the number of bound antibodies An antibody may have any number of fluorescent molecules fluorochromes conjugated to it The number of fluorochromes per antibody is known as the MESF value of the antibody or more commonly the effective fluorochrome to protein ratio or simply the F P ratio The F P ratio of an antibody can usually be obtained from the antibody manufacturer Alternatively the F P ratio may be determined by using the antibody to label a cell or laboratory standard known to bind a specific number of antib
104. he surface charge to the point where Brownian motion brings the particles into contact and they aggregate and stick together in large clumps This electrolyte induced flocculation can be diminished or eliminated by covering the exposed polystyrene surface of the particles with protein more anionic surfactant or nonionic surfactant like Triton X 100 or Tween 20 We suggest the following ways to avoid flocculation during coating 1 AddaSMALL amount of surfactant to particles before exposure to protein buffer solution Particles partially precoated with surfactant are more able to survive electrolyte shock during coating in full strength protein buffer solution Too much surfactant could coat the particles completely and prevent protein adsorption Some people rinse the particles with a dilute solution of protein a few ug mL in lowest possible ionic strength buffer to precoat them Avoid electrolytes use as little as you can and still prevent protein denaturation Keep the particles apart dilute the suspension as much as possible Use as much protein as possible stronger concentration than you might normally add Add particles to protein solution NOT protein to particles Use stirring to promote rapid kinetics get particles diluted and coated quickly n2 SO OB OO ro I IO wa Explanation The idea here is to coat each particle with lots of protein before it encounters another uncoated particle in a hostile high ion
105. ic strength environment The stoichiometry is adjusted so that the particles become protein coated before they become flocculated After coating they will be more electrolyte resistant Index A Ab Ag Binding Reactions sss 35 ABG Values 3 2 rr ertet petet teet 10 AbsOtballCe eene nene tn Be E Er 34 Absorbance Rates cct teet teer 34 Adsorbing Proteles 50 Adsorption 22 26 28 42 45 50 60 61 62 Adsorption Rates sssssesseeeenenennnnnnnns 56 AeroSOlIZatlQIs iconic cites n Pendet 41 44 Affinity Binding 28 Affinity Interactions sisina anas 7 Agglutination esses 34 35 39 45 46 56 Agglutination Assays and Tests 49 54 58 Aggregation sessssseennne 4 16 32 63 64 Alexa FUOD ee erre Hr tees 2 13 anti CD Markers sse 17 anti Human B6eads tcc e 24 anti Human IgG 24 antis MICEODIal c cete edite redeo 62 anti Mouse IgG Beads ssssssssseee 13 Antibody Antigen Concentration esse 56 Antibody Attachment 36 Antibody Binding Capacity sse 4 24 Antibody Coating sse 2 6 Antibody Immobilization eee 62 Antibody Orientation sosnerrrnorrrvorornorrrnerrrvorensorensennnn 42 54 Antibody Quantitation
106. icrospheres Pass Fail Criteria QC Program ViaCheck Viability Standards Carryover Flash Red Fluorophores to the antibody coating Can you address ProMag Bind IT s resistance to harsh conditions ProMag Bind IT microspheres offer a system for highly stable non covalent coating of antibody and we have achieved lengthy stability periods when coated microspheres are stored under normal conditions Though most applications don t call for re use of antibody coated beads after an elution step we have conducted limited testing and found that transitory exposure to low pH 2 8 and high pH 10 elution buffers has little or no effect on mAb coated ProMag Bind IT beads We also found that protein coated Bind IT beads tolerate boiling in 296 SDS for 10 minutes with only a very small loss of protein detected in silver stained gels Ultimately we believe that they should hold up under normal elution steps However we still encourage you to be as kind as you can to them and of course test the specific conditions both to ensure suitability of the immobilization strategy and to determine effects of the process on the mAb itself it probably doesn t matter that the protein remains stably bound if it s being denatured I m trying to coat a biotinylated protein onto your streptavidin coated microspheres however I m having trouble getting the expected amount bound Do you have any ideas of what I could be doing wrong Are there any tips you
107. ider the development of a coating protocol Each contains sample protocols A complete list of TechNotes can be found and TechNotes can be downloaded from our website www bangslabs com What is the value in knowing the brightness of a sample in Molecules of Equivalent Soluble Fluorochrome MESF Equal numbers of fluorochrome molecules do not necessarily have the same brightness Brightness needs to be corrected for changes in extinction coefficient quenching and small spectral shifts MESF units account for most of these environmental corrections For example a cell with a very high expression of a given marker may be labeled with 2 000 000 FITC molecules but due to quenching may exhibit the fluorescence intensity of only 1 500 000 FITC molecules in solution Do you have a suggestion as to what is simple and easy as far as coating antibodies to beads Do you need to know what type of antibody I plan to use The basic bead surface choices for conjugation include non functionalized for adsorption protocols functionalized for covalent coupling and protein coated for affinity binding Each has its benefits which you will need to weigh in the context of your work timetable etc Our TechNote 201 Working with Microspheres provides a discussion of the benefits drawbacks of the different binding strategies You may also wish to view our TechNotes 101 ProActive Microspheres 204 Adsorption to Microspheres and 205 Covalent Couplin
108. idin Biotin Proper orientation when coupling ligands to carboxylate modified microspheres can normally be controlled by optimizing the reaction conditions but believe that the simpler approach is to biotinylate the lectin and attach these to streptavidin coated magnetic microspheres Most commercially available biotinylation kits allow the attachment of biotin by a linker which allows better orientation and less steric hindrance of the ligand at the microsphere surface We absolutely don t know which coupling method would give higher activity but we would bet on the streptavidin SA beads since we load the surface so heavily with SA and it is possible to get more than one biotin lectin bound per molecule of SA Antibody Attachment I am looking for some type of beads that would covalently bind an antibody developed in a chicken IgY I would like to bind the antibody to the beads add Cleavahle Linkers proteins and have the antibody pull out the proteins it recognizes and use the beads to isolate this complex through centrifugation I would then like to break the covalent Denaturation bond by boiling between the antibody and the bead leaving the antibody protein Elution immunoprecipitate Do you carry any type of bead that would allow this covalent bond with an IgY NHS Iminobiotin Bangs Laboratories e Ask The Particle Doctor Page 37 Streptavidin Biotin Divinylbenzene Extreme Temperatures Glass Transition Temperatur
109. ilability of an off the shelf product for the intended separation In these instances further consideration may be given to characteristics of the base particle such as size surface area density composition for tailored handling binding capacity etc Our uniform COMPEL microspheres are well suited for the development of flow cytometric and other bead based assays The low density of the polymer matrix permits binding kinetics that approach those of solution based systems The polymer matrix Bangs Laboratories e Ask The Particle Doctor Page 15 Flow FISH Fluorescent Beads Molecular Biology Quantum MESF Kit References is also amenable to dyeing and the high surface charge allows binding of large amounts of ligand BioMag microparticles are ideal for isolation of cell fractions or purification of target from complex samples Their tremendous surface area and greater density allow rapid and highly efficient capture of the target species For this specific application BioMag is our recommendation BioMag and BioMag Plus are 1 5um high performance superparamagnetic microparticles widely used for the efficient separation of cells and purification of biomolecules Their irregular shape provides a much greater surface area than similarly sized spherical particles resulting in high binding capacities and efficient capture of target with conservative use of particles The high iron oxide content allows for rapid and efficien
110. in or particles I have a problem keeping microspheres dispersed so I keep my 0 8um latex in a 13 sucrose solution in order to keep it from settling Do you have any other suggestions You only need to rotate the bottle occasionally to prevent 0 8um microspheres from settling or if they do settle you can put the bottle on a set of rollers at gt 20 rpm for a few hours to thoroughly resuspend the microspheres Also I m worried that all that sucrose will only encourage microbial growth and attract ants Polystyrene microspheres smaller than 0 5um will never settle in H O Brownian motion or diffusivity is greater than settling velocity The best conditions for storage of microspheres 0 5um are 1 Keep with surfactant to maintain colloidal stability 2 Add antimicrobials like sodium azide or thimerosal 3 Put them on rollers at low speed 5 8 rpm to keep them suspended 4 Keep temperature at 4 8 C to suppress growth of microorganisms Why can t I keep my 0 3um coated microspheres dispersed after have coated them Small singly dispersed PS microspheres will neversettle see question above even after they are protein coated However microspheres can easily become clumped during cleaning especially in a centrifuge or during coating with protein And clumps of microspheres like bunches of grapes will settle You must ensure that the microspheres in your product are truly singly dispersed Check with a micr
111. ing esee 23 37 PEGS s eee UL NEAL LT UMS 30 Peptide Binding sse 20 Percent Solids Determination sss 36 PETIAC 5 34 He 28 62 Phage Yeast Library sess 18 Phagocytosis d ci dtt ee nen RR RES 60 Photobleaching nennen 27 PIM 44 Polyclonal Antibodies sese 61 PolyLink Protein Coupling Kit sss 7 14 18 aki 5 Polystyrene Microspheres sse 9 ProAgtiNeQ ses erase 32 33 34 38 39 42 ProMag cit cna eese tiit etre tette cerit deindc 2 12 18 ProMag Bind IT M rnmsnerovorvovovoverevrsvererevesveverenesssvsvenerssvsnenen 3 ProtenAvu 2easta NN 7 Protein Adsorption essence 30 51 Protein A Microspheres esses 4 14 42 Protein Binding oeorerrnrorvornenrorrrsvanrnrrerornrrverrornenvennen 53 59 Protein Coating ssorororerorvsvorsvonerrsvovererrsvsverevesvsvsvenesrsvsvener 47 Protein G Microspheres seen 4 Protein L Microspheres sse 42 Protein LoadifiQ ccrte trem 33 Protein Quantification ssseeeennene 34 Purifying Samples sse 14 Q OGO M oes Men ie are eae een en 17 OG PROQEATL s ticis eee eset nent hes nena dnce cen eco 3 QC Windows sse 17 Quality Assurance
112. ing reactants and concentrating in one campaign without changing the filter set up only one pot gets dirty It may be difficult to make easily redispersible dry beads this small without clumping them You might try aerosolizing them and drying the spray of droplets sized so that there is 0 1 bead per drop Then when the drops dry there is only one bead so two beads cannot be pulled together by surface tension during drying It may also be possible to freeze dry the beads from dilute suspension so as to produce a free flowing powder of single particles Either drying process may require addition of surfactants blockers like proteins and sugars If you can find a way to work with a liquid particle suspension it will certainly be easier Step 2 Some folks use a shorter time Step 3 You mention diluting the reaction with a different buffer If the activation Step 2 is done at acidic pH then be sure to add enough of the higher pH buffer Step 3 to get the pH above neutral so that the primary amines on your ligand are in the correct form for the covalent reaction You might try pH adjusting the final solution to achieve this We like the idea of quenching with ethanolamine to use up remaining active esters and to produce a hydrophilic surface which is less likely to adsorb unwanted protein Step 4 sounds good As to your Main Question We think that surfactant can be used to prevent unwanted binding of proteins after covalent
113. ions Parking Area Surface Charge Density Titration Data COOH Modified Beads Covalent Coupling One Step vs Two Step Coupling Q Are you able to supply information about the number of carboxylic acid groups on the surface of the beads We do titrate our carboxylated beads This information is provided on our website www bangslabs com and on Certificates of Analysis where appropriate The surface charge density in your catalog is mentioned in terms of microequivalents of acid or base per gram of microspheres What does that mean in terms of number of electronic charges or coulombs per particle A We take an aliquot of ion exchange or cross flow filtration cleaned microspheres in suspension measure solids by weight loss on drying and calculate the weight of microspheres to be titrated Then we titrate this volume of microspheres with acid or base of known concentration until the acid base equivalence point is determined conductimetrically or potentiometrically from this we calculate microequivalents of COOH groups for example per gram of particles There are 6 022 x 10 molecules mole of 6 022 x 10 charged groups microequivalent Coulombs Conversions There are 96490 Coulombs equivalent of COOH for example Therefore there are 6 022 x 10 96490 6 241 x 10 8 molecules C or 1 602 x 10 C molecule For a sphere of diameter d the surface area A rrd volume V md 6 0 5236 d and mass M Vp where
114. issolved in some solvent maybe DMF and measured in a spectrophotometer at a wavelength sensitive to polystyrene I do not know of such a method I m only composing at the keyboard Bangs Laboratories e Ask The Particle Doctor Page 31 Dilution Suspending Dry Microspheres DNA Adsorption References Silica Adsorption Silica Microspheres Carboxylated Beads Parking Area Surface Titration Value Q I have purchased BLI microspheres both dry and in suspension Please advise how to dilute a suspension of microspheres and how to suspend the powdered microspheres especially what solutions are needed Microspheres may be diluted using the buffer they come in often DI water or the buffer of choice following centrifugation or other separation method see our TechNote 203 Washing Microspheres for more information regarding separation methods Additional buffer may be added directly to the suspension or may be added to the pellet following centrifugation To suspend dry microspheres add the buffer of choice and mix e g using an end over end mixer roller vortexter or sonicator very carefully Surfactant may be added if aggregation is observed see our TechNote 202 Microsphere Aggregation for details The duration of the mixing process will depend upon the size and amount of microspheres i e anywhere from a few minutes to a few hours of mixing may be required You may wish to periodically check the progress
115. ith a monolayer of COOH groups This number comes from the packing density for a close packed monolayer of fatty acids at an air water interface The number for sulfate groups would be about the same primary amino groups might be smaller One envisions a model where all the microspheres are the same diameter and the charged groups are neatly arranged closely packed on the surface of the microspheres This is strictly true only for sulfate modified microspheres and for certain COOH modified microspheres which have charged groups only at the ends of the polymer chains Most of our COOH microspheres are made by copolymerizing a small portion of acrylic acid with styrene S AA In this case the COOH groups are on random polymer chains which will tend to arrange themselves with the hydrophilic COOH groups in the aqueous phase and at the microsphere surfaces The COOH groups are certainly not arranged as a neat monolayer here but probably exist as hydrophilic chains attached at one or both ends and extending out into the aqueous phase rather like tennis ball fuzz The 20 2 COOH group rule can very easily be violated here of course If particles with non uniform size like our magnetic particles are considered then the model based upon a calculation on spheres of one diameter will fail again due to errors in estimating surface area per gram With S AA microspheres often only about half of the acid which is added actually appears on the particles
116. k if you add the particles to the protein solution rather than protein to particles order of addition is often important in colloid chemistry How can covalently bind more IgG on your carboxylate modified beads used 3 5 g IgG liter and didn t see much difference in protein concentration before and after coupling A Here are a few ideas for how to load on more protein 1 2 3 The particles should have plenty of carboxylic acid groups on their surfaces usually about a monomolecular layer of acid groups covers the particles Our particles also have sodium dodecyl sulfate added to ensure uniform dispersion This surfactant will be adsorbed onto the particles We recommend that you clean the particles and remove surfactant to make more room on the surface for the protein For particles with mean diameters of 1um the specific surface area m g is 6 d where d is diameter in um Thus 1pm particles have 6 m g The maximum adsorption rate of IgG onto polystyrene is 2 3 mg m this is a monolayer of complete coverage of protein Therefore one can put on 6 x 3 18mg of IgG g of 1um particles If you used 1mg IgG 10mg of particles you should have 5 fold excess over monolayer amount 0 18mg protein 10mg of particles or plenty to ensure complete coverage Equilibrium adsorption of BSA onto polystyrene occurs at 0 1g BSA liter One must add enough protein to achieve monolayer coverage and with as little as 0 1 protein liter
117. le aggregation is an interesting problem We have been urging attention to possible aggregation for customers who prefer to clean the particles by centrifugation decantation redispersion Also a sensitive detection method can be the basis for Latex ImmunoAssay LIA can suggest several methods in order of increasing sophistication 1 If the particles are large enough gt 0 5um they may be observed in the light microscope and approximate counts of singles doublets etc may be made This method must be used with caution because the necessity for sample dilution before observation may introduce an artifact the act of dilution may cause or eliminate the particle aggregation you are trying to observe Of course this method will not work for smaller particles Spectrophotometers can be used to monitor particle aggregation The light scattered by single small particles will change if the same number or weight of particles are partially aggregated The scattered light may be read as absorbance on any spectrophotometer If absorbance changes with time or differs from batch to batch one can make inferences and conclusions about the aggregation state However it may be difficult to quantify the exact numbers of doublets triplets etc Again dilution may cause changes in the state of aggregation Nephelometers may be used similarly by reading scattered light directly Usually these instruments have a fixed angle often 90 between the det
118. ll D J Newman E A Medcalf C P Price 1991 Particle enhanced turbidimetric immunoassay of sex hormone binding globulin in serum Clin Chem 37 4 527 531 However we worked with a customer who says that for these nephelometric assays the surface groups are more important than the refractive index of the core polymer Meanwhile some other work showed that lower refractive index polymers would be useful Amiral J M Migaud 1991 Development and applications of a new photometric method for fast and sensitive immunoassays Europ Clin Lab 10 28 We have many sizes of polymethyl methacrylate PMMA particles with lower refractive index for you to try if you like Q Gan I check surfactant concentration on incoming lots of latex by measuring surface tension You can use surface tension to compare incoming lots of latex if they contain sulfate or sulfonate surfactants If however the surfactant is a fatty acid sale you should check pH also Sulfate surfactant molecules will normally all be in the SO form so surface tension will probably be independent of pH at pH 5 Fatty acid surfactants with pKa s 55 2 are more sensitive to pH and will have COOH as well as COO species present at higher pH each contributing differently to surface tension So always measure surface tension at the same pH gt 11 recommended Q What is this parking area Notan employee perk it refers to the area per molecule for titrated acid or other
119. longer answer is that the one step method involves combining microspheres water soluble carbodiimide WSC and protein in a neutral pH buffer all at once The advantage is that it is only one step only one pot gets dirty Bangs Laboratories e Ask The Particle Doctor Page 49 Microsphere Dispersion Storage Conditions Sucrose Agglutination Tests Clumping Coated Microspheres Reproducing Clump Sizes Settling One disadvantage to one step is that the pH normally used is a compromise between the ideal pH for both halves of the coupling reaction The BIG disadvantage is that the WSC is indiscriminate it can crosslink protein as well as bind it to the particles and it is possible to bind everything together protein crosslinked protein and microspheres You can easily form clumps of particles which are covalently bound together by crosslinked protein The clump sizes are uncontrollable and a wide distribution of clump sizes can be formed It may be possible to do this coupling reaction successfully in one pot But if folds are successful we don t know about it because they don t call to say Eureka We hear about the problems if any so we are constantly teaching ways to avoid the clumping problem This is easier than breaking clumps once formed because Breaking up is hard to do Neil Sedaka The two step method involves reaction of microspheres with WSC at a low pH where the carboxylate groups are in the COOH form t
120. mall cells that noon IR will be dimmer due to their size Examples include telomere length ids determination and some cell surface markers e g CD34 Medium level kits are used for many types of analyses and nicely span the range of typical cell samples Common analyses include those for ej gent ent CD38 Plasma Cells many surface markers including CDA CD8 High level kits are often caus used for cells with very high expression or high autofluorescence e g analysis of tumor cells J If you re still wondering which is best suited for your assay you might start with the mid level kit which overlaps areas of the low and high kits Catalog Code Description 824 Quantum FITC low level 824p Quantum FITC low level premixed 825 Quantum FITC high level 825p Quantum FITC high level premixed 826 Quantum FITC medium level 826p Quantum FITC medium level premixed am gathering information on ZAP 70 staining of CCL samples and would like information on which quantification product to select have spoken to other labs using ABC binding beads that they are staining with ZAP 70 antibody Is this preferable over a particle using MESF What advantages are there if any of one over the other Both Quantum MESF and Quantum Simply Cellular QSC kits would be suitable for use although there are some differences that may lead you to prefer one approach over the other Beginning with similarities
121. n Exchange A IX is a quantitative fast one step process It removes all ionic species except those stuck on the particles like SO and COO and converts all the particle groups to the neutral species SO H and COOH XF filtration removes all water soluble material Cleaned particles are usually about half charged with half of the sulfate and carboxylate surface groups in the SO H and COOH forms The IX cleaned surfaces thus have a lower charge and the particles are not as stable XF filtration takes longer since it depends upon passive desorption of surfactants from the surface Overall would choose XF first Bangs Laboratories e Ask The Particle Doctor Page 56 Surfactants for Protein Q Which surfactants are better to use with protein coated particles Adsorption Rates Equations Agglutination Antibody Antigen Concentration Binding DNA Binding Oligonucleotides DNA Adsorption Streptavidin Biotinylated DNA Nonionics probably In studies where high levels of surfactant were added to try to remove protein nonionics could only remove 60 of adsorbed protein while anionics removed 80 To detect a certain virus by latex agglutination we tried to adsorb polyclonal and monoclonal antibodies onto 0 8um and 2 5um polystyrene particles First we washed 50mg of particles several times with buffer Then we resuspended them in 4mL of 0 3mg of Ab mL buffer solution and incubated overnight at room tem
122. n developing a particle enhanced turbidimetric immunoassay PETIA The monoclonal antibodies mAb s we have are only suitable if they are coated to latex beads What type of latex beads PETIA would you suggest ProActive First try some 100nm PS beads with direct adsorption of mAb plus adsorption of blocker to fill in empty spaces between mAb See TechNote 204 Adsorption to Microspheres Some mAb s do not adsorb well Turbidimetric Assays directly and they must be covalently bound For that try COOH modified beads of the same size See TechNote 205 Covalent Coupling To get a product more quickly try some of our goat anti mouse GAM coated ProActive beads Our GAM was selected and tested for binding to almost all common mAb s Settled Microspheres had to use twice the amount of antibody Ab to sensitize the microspheres in the last 10 of the bottle What went wrong Suspension Dispersity A It could be several things 1 Incomplete dispersion of any settled microsphere solids could yield a more concentrated latex by the end of the bottle and more solids would require more Ab If solids have settled to the bottom of the bottle then before every aliquot is removed we recommend several hours of rolling the bottle to thoroughly resuspend the beads and to ensure that any clumps are broken up so you will have singly dispersed beads Rolling action avoids foaming which can result from shaking 2 Beads can become concen
123. n happen with any particles unless they are very hydrophilic and have protein bound covalently or unless they are well blocked every hydrophobic site filled with blocker molecules or surfactant Here are some possible solutions 1 can imagine proteins in urine adsorbing on latex we call it nonspecific binding or NSB to cause nonspecific agglutination Alternatively these proteins Try quenching any remaining reactive groups and create hydrophilic surface groups which will not adsorb protein With your CH CI particles after you have bound your protein you might quench unreacted chloromethyl groups with Bangs Laboratories e Ask The Particle Doctor Page 46 References Urine Based Tests Agglutination Clumping Surfactant Desorption of Protein Storage Surfactant Binding Polypeptides References Clumping ethanolamine or diethanolamine converting these hydrophobic chloromethyl groups to hydrophilic non adsorptive hydroxyl groups After binding IgG or other desired protein on the microsphere surface use plenty of blocker BSA casein Tween 20 etc to fill up uncovered sites which might bind that urine protein You can try more hydrophilic COOH modified microspheres These can adsorb proteins too but probably not as much as the more hydrophobic S VBC particles Blockers would be appropriate here too After protein coupling you also might want to try quenching any remaining unreacted groups again wi
124. n the ABC value equals the number of surface receptors QSC kits are available for mouse rat or human monoclonal primary antibodies Catalog Description 488 Quantum Alexa Fluor 488 MESF 647 Quantum Alexa Fluor 647 MESF 815 Quantum Simply Cellular anti Mouse IgG 816 Quantum Simply Cellular anti Human IgG 817 Quantum Simply Cellular anti Rat IgG Sterilizing Q am hoping to find sterile microsphere preparations for use in an animal study Do you offer sterile Microspheres suspensions A While we don t offer sterile microsphere preparations as standard products microspheres may be sterilized through irradiation or 70 alcohol incubation It s also possible to conduct stringent washes in sterile medium or pasteurization if the need for a sterile suspension isn t absolute Our Product Data Sheet 726 Decontaminating Polystyrene Microspheres offers a number of decontamination protocols and may be downloaded from the Technical Literature section of our website www bangslabs com literature pds BioMag Q I m in need of magnetic microspheres that can be coated with antibody What options do you have COMPEL We offer three different lines of superparamagnetic microspheres ProMag BioMag and COMPEL which allow us to uniquely address a wide range of applications from cell separations and Magnetic Microspheres immunoassays to flow cytometric suspension arrays While each type can be used for varied purp
125. n the antigen hapten then you should be able to put one Ab on the dyed particles and put the second capture Ab on the strip at the capture zone Then if a sample contains antigen hapten it should form a sandwich between the dyed particles and the capture Ab Bangs Laboratories e Ask The Particle Doctor Page 55 Centrifugation Q What s wrong with your 20nm polystyrene particles can t spin them down in my centrifuge Cross Flow Filtration Particles are OK Polystyrene particles of density 1 05 g mL will settle in pure water p 1 00 g mL if Small Microspheres you can apply enough acceleration G forces For 20nm particles it will require gt 3 x 10 G s really to achieve 10 cm hr settling velocity And if you do spin them down they will be very difficult to resuspend Try cleaning the particles with cross flow filtration or a Sephadex G 25 column See TechNote 203 Centrifugation Q How do you calculate the G forces required to achieve a settling velocity of 10cm hr Equations We calculate it from Stokes law formula for settling velocity of spheres v 5 448 x 10 p 1 d where Settling Rates v cm sec is maximum settling velocity at normal acceleration 1G for a sphere of diameter d um and density p g mL in water We divide the desired settling velocity e g 10 3600 cm sec by v to arrive at Stokes Law the G s necessary to achieve the settling rate Loss on Drying Q How can measure low particle
126. nce to supply any particles you need We offer free technical service and literature to customers and prospective customers with questions which we can answer by phone fax or email See our collection of TechNotes www bangslabs com We can coat polystyrene spheres with your antibody of choice on a custom basis contact our Customer Service Department at info bangslabs com to request an official quote We have colleagues who can also help you scale up for your in house preparation Ask us to supply their names and addresses Q How can I bind protein to your particles via a disulfide bond S Wong CRC Press 1991 especially pp 58 amp 61 highly recommended by us as an excellent reference for coupling things to particles via a wide variety of chemistries and from Pierce Chemical Company s excellent booklet on SPDP First react primary amino modified particles plain or magnetic with SPDP N succinimidyl 3 2 pyridyldithio propionate a heterobifunctional cleavable cross linker or related analogs The reaction proceeds via the NHS ester end Then react the 2 pyridyl disulfide activated particles with cysteine groups on protein or peptide The We were not familiar with this chemistry but found the answer with some help from the book Chemistry of Protein Conjugation and Cross Linking by Shan Bangs Laboratories e Ask The Particle Doctor Page 54 SH thiol group on the cysteine reacts with the disulfide group on the p
127. nd inside the tube Q What s the matter with your particles could only put 200 300ng of protein per cm of particle surface Congratulations You achieved maximum coverage of protein on the particles The best one can do is a monolayer of protein molecules which is 3 mg m 2300 ng cm See our TechNotes 204 or 205 for details on calculating expected coverage We can do an agglutination test with bacteria antibody Could we mix bacteria particles antibody It sounds like a good idea Particles would add bulk to the test to enhance visibility and sensitivity One way would be to put Ab to bacteria cell surface antigens on the particles then any added bacteria in a sample would cause coagglutination My dyed particles don t have any get up and go How do put them on a membrane and prevent them from sticking permanently Covalently bind protein to the particles Then add lots of surfactant to them and to the membrane to prevent the particles from sticking to nitrocellulose or any Bangs Laboratories e Ask The Particle Doctor Page 59 Strip Tests Strip Tests Blockers Nonspecific Binding Protein Binding Surfactant Water Soluble Carbodiimide Dilute Suspensions Particle Concentration Solids Content Turbidity other hydrophobic membranes Pretreatment of the membrane with substances that can easily be rehydrated and maintain a small space between the beads and the membrane e g sucrose solution ma
128. ne particles between 0 01pm and 0 49um that are dyed blue On each of these COAs the catalog code is DS02B The second number on the COA is the Bangs or BLI Lot Number e g 0193 3283 amp 0248 This number is specific for a particular lot of particles So each of these three particles have the same catalog code but different Bangs Lot Numbers specific for each lot of particles When a new product in that range is made for you it will have the same catalog code DS02B but a new BLI Lot Number We also create OEM Catalog Numbers for regular customers Once we receive specifications on a product from you we can apply an OEM Catalog Code that will encompass any lot that meets those specifications When ordering the product you will simply have to give the OEM Catalog Number We will start this process as soon as we get a copy of your specifications for the different products Q I got very low binding of my biotinylated ligand to your SuperAvidin coated beads using a buffer with 196 BSA to prevent unwanted NSB What s wrong with your microspheres Oh great You have a problem and it s our fault But seriously you may have used too much BSA We found that 0 05 BSA 1 20th of what you mentioned in the storage buffer is enough For more blocking try adding 0 01 Tween 20 it worked great for blocking NSB of biotinylated acridinium ester in a binding study we published Q What can you advise us about using sonication to redisperse micr
129. ngth and solvent resistance The microspheres will not swell in several solvents like ethanol methanol normal alkanes and others We can send a list The PS particles will dissolve to some degree in many other organic solvents like Bangs Laboratories e Ask The Particle Doctor Page 44 benzene toluene some esters higher ketones methylene chloride and other chlorinated solvents If the particles are crosslinked with divinyl benzene DVB they will only swell in these same solvents The degree of swelling depends on the solvent and is inversely related to the degree of crosslinking i e more DVB less swelling Changing Solvents Q How do I change phase to the solvent want Organic Solvents To change from water to solvent you can just let the microspheres settle by gravity or centrifuge decant the supernatant add a solvent which is miscible with the previous liquid water like methanol ethanol my favorite or iso propanol and redisperse the beads Repeat this until you have the beads in the liquid you want Changing Solvents Q Will centrifugation or gravity work for small 0 1um beads which don t settle fast Cross Flow Filtration Well it certainly will be harder longer to spin down these small beads and redisperse them to single Organic Solvents particle suspensions By using cross flow filtration equipment it is now possible to change phase without causing the beads ever to get clumped or to touch each other
130. nt protocol suggests blocking with BSA and non ionic surfactant in the weight ratio of 20 1 1 BSA 0 0596 Tween 20 See Jenkins S H H B Halsall W R Heineman 1990 The use of ion pairing reagents to reduce nonspecific adsorption in a solid phase electrochemical enzyme immunoassay J Clin Immunoassay 13 2 99 104 One can also covalently couple the Mc Ab s to surface modified particles to ensure their firm binding Q What do we do if the particles are contaminated Bangs Laboratories e Ask The Particle Doctor Page 62 pH A We don t usually have problems with bioburden or wildlife In many of our particles the combination of the surfactant used fatty acid and pH gt 8 hinders microbial growth We also store our particles at 4 C after they are made Nevertheless particles can become contaminated after repeated entry into the bottle At the request of some customers we have added an antimicrobial either sodium azide or thimerosal sodium ethylmercurithio salicylate to some particles If you have a contamination problem and don t want to add an antimicrobial try adding base to pH 11 and store for about two weeks COOH Modified Beads Q How should we bind our protein to carboxylic acid modified particles Covalent Coupling Try the two step method First react the particles with water soluble carbodiimide WSC at acid pH EDAC Coupling 4 6 Clean to remove unbound WSC Second react WSC activated particles with protein at
131. o want to attach a biotinylated oligo to streptavidin microspheres but don t know how much to include in the reaction would also like to determine the amount of biotinylated oligo that is actually bound What Quantum MESF Kit method can use Streptavidin Coated The binding ability of our streptavidin coated microspheres is assessed through the binding of biotin FITC Microspheres The binding capacity is reported on the Certificate of Analysis in terms of ug biotin FITC mg microspheres This value may be used to estimate the capacity for your biotinylated oligo For a basic attachment protocol with recommendations for oligo concentration see our Product Data Sheet 714 Binding Biotinylated DNA to Streptavidin coated Microspheres Titration of the Binding Capacity of Biotinylated After coating there are a number of methods for Oligo d T 20 Coupled to 1mg ProActive Streptavidin determining the amount of immobilized oligo You au d may read the 0D260 280 of the supernatant following the binding reaction or use a fluorescent nucleic acid stain for single stranded DNA If the oligonucleotide or its target is labeled with a fluorochrome flow cytometric analysis may be utilized with an appropriate Quantum MESF kit e g Catalog Code 826 Quantum MESF FITC to orn quantitate fluorescence intensity of the bound Bangs oligonucleotide on the streptavidin microspheres Mean Fluorescent Amount of 5 Biotinylated Oligo d T 20 FITC u
132. o form an activated intermediate followed by cleaning to remove unreacted WSC Note that the active intermediate is unstable and immediately begins to hydrolyze Cleaning should be rapid to minimize hydrolysis maximize of active sites that will bind protein Then protein is added and the pH adjusted to the basic side pH 8 so that the amino groups are in the NH form Again after coupling protein the microspheres are cleaned to remove unbound material While the two step process indeed takes more steps we think you have better control of each step and the whole process and can easily avoid forming microsphere clumps Call us for coupling protocols and tips for trouble free covalent binding One suggestion is to use crossflow systems to do all coupling and cleaning steps in the same reactor If you insist on doing a simplified one pot one pH process then we would suggest the following 1 5 step method 1 Carefully calculate how many COOH groups you need to activate dry weight of particles x millequivalents of COOH gram 2 Add only enough WSC to activate all surface groups may require some excess 3 Let this first part of the reaction proceed for perhaps 1 2 hour at room temperature 4 Calculate how much protein you intend to bind to the particles 5 Add only enough protein to saturate the surface Such a process should allow covalent binding of protein to particles without any extra WSC or protein to permit crosslinking of prote
133. oated Microspheres COOH Modified Beads Surfactant Free Washing Beads Water Soluble Polymer WSP COOH Modified Beads Titration Data Agglutination Chemiluminescent Based ELISA separated magnetically or by centrifugation By this positive selection excess dye terminator is left in solution and the sequences of interest are captured on the beads The captured strands can then be eluted from the bead using 95 formamide at 90 C The isolated sequences can now be run on the gel See TechNote 302 Hint Be sure to denature the strands and put the vessel immediately on ice prior to bead capture of the strands Capture of double stranded DNA may be 50 less efficient Q To recover concentrate bacteria we wish to coat magnetic particles with our rabbit anti bacteria antibodies in serum form We are thinking of your ProActive magnetic beads coated with goat anti rabbit antibody Could you please suggest the right beads plus instructions for using them We have goat anti mouse GAM IgG coated magnetic microspheres but no goat anti rabbit GAR beads yet To use GAM beads you would need to bind mouse anti rabbit MAR antibodies to the GAM then bind your rabbit anti bacteria RAB antibodies Are you confused yet We also offer streptavidin coated microspheres If you biotinylate your antibodies you could attach these in a one step coupling reaction with a bond strength nearing that of a covalent bond For this approa
134. odies and then comparing the MESF value of the labeled sample to its binding capacity Once the F P ratio is obtained it is easy to determine the number of antibodies bound to the cell simply divide the MESF number by the F P ratio MESF Values Biotin Binding Q Will streptavidin biotin binding be stable enough to survive PCR thermocycling PCR Thermocycling Short answer Several customers have reported that our SA beads work well for PCR work Very long answer Here are some references from my exhausting but not exhaustive literature search regarding References stability of strept avidin and the strept avidin biotin complex Streptavidin 1 Bayer E A et al 1996 Sodium dodecyl sulfate polyacrylamide gel electrophoretic method for assessing the quaternary state and comparative thermostability of avidin and streptavidin Electrophoresis 17 8 1319 1324 PubMed 8874057 On heating in the absence of biotin the quaternary structure of streptavidin is more stable than that of avidin 2 Wang C et al 1996 Influence of the carbohydrate moiety on the stability of glycoproteins Biochemistry 35 23 7299 7307 PubMed 8652506 Apparently deglycosylated avidin SuperAvidin NeutrAvidin is less stable than avidin the carbohydrate moities conferring greater stability to the protein If unbound streptavidin is more heat stable than avidin then bound streptavidin should be more stable than bound deglycosylated avidins although the
135. oing cell purification depletion studies I m trying to tag and remove certain cells with your magnetic particles How do stop my cells from engulfing or eating the magnetic particles Dr Ramani from Transmed Biotech says there are two ways of stopping cells from eating particles 1 lower the temperature or 2 add some azide not enough to kill them just spoil their appetites Also remember that the more hydrophobic the particle the more likely it will be phagocytosed Q How can I use fluorescent particles in immunoassays A We can think of at least two ways Flow method Imagine that the particles are put through a capillary and individual particle fluorescence is measured The fluorescence of the dye is excited at one wavelength and measured at another wavelength You should get light flashes of one intensity for single particles As particles start to agglutinate you should get flashes of higher intensity as doublets triplets etc pass by Quenching method Now imagine that all the particles are measured at once dilutely dispersed in a tube If they are agglutinated or clumped then some of the fluorescence will be absorbed or scattered and lost and the signal will diminish on agglutination See also the Gosling paper cited below Gosling James P 1990 A decade of development in immunological methodology Clin Chem 36 8 1408 1427 Filter Method If single particles pass through a filter but agglutinated particles ar
136. omputer will be able to search your purchases and offer you the files appropriate for the bead kits you have purchased This way we can be sure that you always get the correct files without your having to select the right one from a long list of files Q How do coat your beads with lectins such as wheat germ agglutinin WGA For the immobilization of WGA a number of strategies may be utilized For one common method see Ertl B F Heigl M Wirth F Gabor 2000 Lectin mediated bioadhesion preparation stability and caco 2 binding of wheat germ agglutinin functionalized Poly D L lactic co glycolic acid microspheres J Drug Target 8 3 173 184 They used the carbodiimide N hydroxysuccinimide method which is a fairly standard method for coupling ligands to COOH functionalized microspheres see also our TechNote 205 In any case lectins will have COOH and NH termini that could be utilized for immobilization WGA also possesses numerous cysteine residues which could be utilized for the formation of disulfide bonds i e a bead could be modified using a heterobifunctional crosslinker that will react with amines bead and sulfhydryls WGA This is a strategy similar to others presented in TechNote 205 EDAC is a zero length crosslinker for joining COOH and NH groups glutaraldehyde is a homobifunctional crosslinker utilized for binding NH groups Visit our sister company Polysciences at www polysciences com for glutaraldehyde and
137. one can maintain equilibrium and keep the protein on the particles Your 3 5mg of IgG mL 3 5 g L solution is much more concentrated than necessary 0 you may see no detectable difference in absorbance of a solution before and after coupling The recipes in our Covalent Coupling TechNote TechNote 205 should be used with your own good judgment Feel free to change buffers pH s and concentrations of ingredients to optimize conditions for your specific situation You might try to simply adsorbthe IgG onto the particles just to see if you get any more protein onto the particles by adsorption This would tell you if the coupling chemistry is at fault somehow 1 Do you have any polystyrene beads with DNA covalently attached to the surface notice that you sell PS beads with certain functional groups proteins and fluorescent labels 2 DNA probes PCR was listed as one of the applications of the beads Does it mean that a DNA primer is attached to the surface of the bead or just via electrostatic interaction via a functionalized bead Please let me know so I can decide what kind of beads to buy and if any chemical modification has to be performed on the beads silica beads are useful for nonspecific attachment of DNA Reference 1 Volume 13 2 June 2000 2 You can electrostatically or covalently attach DNA to our carboxyl modified polystyrene microspheres Reference 2 Volume 13 72 June 2000 and TechNote 205 3 You can biotinylate the D
138. or that approach then perhaps you can try chemiluminescent based ELISA using magnetic microspheres We can offer magnetic beads with whatever ProActive coating you want If you want to do it yourself then we can always supply bare mag beads and we ll help you all the way to a successful coating of these Q How can I bind DNA to your silica beads non covalently W R Boom seems to use salt and chaotropic agents to make the liquid phase so nasty Let s get out of this place that the nucleic acids are more comfortable on the particle surface We don t think that there is any specific interaction between the silica and nucleic acid See our TechNote 104 and our TechNote 302 for ideas on the reversal of silica charge to for DNA pick up See also Melzak K A C S Sherwood R F B Turner C A Haynes 1996 J Col Interface Sci 181 635 644 Q tried three different lots of your CML COOH modified latex microspheres All three lots A B amp C were about the same size and had the same amount of acid on the surface Without cleaning them adsorbed protein washed away excess protein with buffer and added EDAC to covalently link the protein onto the surface All three beads adsorbed the same amount of protein and had the same amount bound after covalent coupling but C had zero reaction with the antigen So what happened While they were all the same diameter and acid concentration the three beads you ordered were m
139. oratories e Ask The Particle Doctor Page 4 Glacial Blue Antibody Binding Capacity Antibody Weights Quantum Simply Cellular Certified Blank Protein A Microspheres Protein G Microspheres Simply Cellular anti Mouse Compensation Standard To a greater or lesser extent broad emission or fluorescence carryover into other unintended regions of the spectrum is characteristic of all fluorophores It is particularly evident with highly sensitive instruments such as confocal microscopes and flow cytometers It probably wouldn t be apparent with a standard fluorescence microscope and if you ve got one of those black lights well you have nothing to fear as you won t see anything except your white T shirt and tennies Did someone say dance party Remedies might include changing the filter sets for example to more stringent bandpasses or the fluorophore itself Fluorophores in the middle of the spectrum e g with blue or green excitation maxima tend to have significant carryover into regions that have historically been used in detection of reporters in bioassays e g green orange red It s not that the carryover is more severe with fluorophores inthis region rather it s occurring at an inconvenient place We have observed thata number of UV Violet e g Glacial Blue and Red e g Flash Red fluorophores tend to have little carryover into the green and orange and might be better suited to your study if you nee
140. orbed coatings in general and particularly where beads will be stored in suspension shelf life should be considered Once you ve had a chance to consider the specific structure of the LPS molecule and factors such as required stability and development time frame we can provide references associated with a fitting coating strategy I m familiar with the use of polymer beads in flow cytometry both as instrument QC and set up standards and for bead based assays However inlooking through Bangs Laboratories e Ask The Particle Doctor Page 6 References Silica Microspheres Fluorescent Beads Mounting Mediums Antibody Coating Carboxylated Beads Coupling Process your catalog see that while you carry silica microspheres you don t offer silica bead standards for flow Out of curiosity are there any known applications for silica beads in flow cytometry A We are delighted that you asked By happy coincidence flow cytometry and silica microspheres are two of our favorite things Uncanny isn t it Because of silica s unique optical and physical properties e g low autofluorescence and hydrophilic surface it has been used as an alternative to polystyrene for certain applications in flow cytometry For example Silica exhibits less autofluorescence than does polymer when excited with a UV or violet laser In fact we featured NH modified silica in our newsletter as a potential substrate for the binding of
141. oscope or particle measuring instrument for change in mean diameter and distribution during Bangs Laboratories e Ask The Particle Doctor Page 50 Adsorbing Protein Adsorption Coating Beads Dialysis Small Beads Surfactant Washing Beads your processing Unfortunately this clumping can make a big difference in the performance of a test or assay Single microspheres will migrate in a strip test much faster than clumped microspheres which may not move at all Agglutination based tests which have preclumped microspheres will have very different performance sensitivity than those using single microspheres The worst part of this problem is the extreme difficulty impossibility of reproducing the needed clump size One might need to use elaborate processes like a special stirring protocol to make the right clump size Or use elaborate schemes involving mechanical shearing or ultrasonication to try to reproduce the correct clump size by breaking very large clumps down to just the right size If you think that you may have a problem with a test or assay which uses and depends on clumped microspheres call us You perhaps inherited a project where the product was unintentionally designed around clumped microspheres If these products were now made with singly dispersed microspheres they might not work at all We think it s much better to design your product to use single microspheres and we will gladly help you choose the right size and
142. oses we do find that each type offers particular advantages to certain applications As highly uniform microspheres with high ProMag coating capacities and rapid separation rates ProMag 1pm 3um offer significant benefits for automated immunoassays BioMag are 1 5um high performance microparticles that are widely used for the efficient separation of cells and purification of biomolecules The irregular morphology provides tremendous surface area resulting in high binding capacities and efficient capture of target with conservative use of particles COMPEL are highly uniform polymer based microspheres 3um 6um 8um that have size and autofluorescence profiles that are ideal for applications in flow cytometry The polymer matrix is conducive to dyeing and standard red and green fluorescent versions are available Of course we are always prone to waxing philosophic about our microspheres and you can find images and more on the characteristics and benefits of each type in TechNotes 102 and 102A on magnetic microparticles These TechNotes can be found at www bangslabs com literature technotes Antibody Coating I m interested in your new pre activated ProMag Bind IT microspheres however my process includes an elution step that I m afraid will be detrimental Bangs Laboratories e Ask The Particle Doctor Page 3 ProMag Bind IT Binding Capacity Binding Efficiencies Biotinylated Proteins Streptavidin Coated M
143. ospheres after centrifugation You can use gentle sonication to redisperse microspheres Use only enough sonication to redisperse beads to a smooth dispersion but not enough to heat your suspension An ultrasound bath would be preferred because there is less chance of using too much energy and no chance of contamination from a probe We have not heard of any problems with sonication of protein coated beads but we would exercise caution especially if protein is adsorbed or coupled via long tethers which could break if the protein bead link is whipped around too much By the way a customer told us that our SuperAvidin coated beads show no losses of binding capacity after sonication have some of your larger beads 100 200um but they are hydrophobic How can get hydrophilic beads this size found some references for creating active surface groups on the beads one was in your 1984 book Uniform Latex Particles but these references mentioned reacting the beads with fuming red nitric acid and sulfuric acid followed by reducing agents to yield amino groups I m not a chemist and all those acids sound scary and I only want them to be hydrophilic 1 You are right Concentrated acids like glacial acetic acid and fuming red nitric acid are very nasty to work with Be very careful You should get some Bangs Laboratories e Ask The Particle Doctor Page 41 help from a chemist at your place You will need a plastic apron
144. otein A G Try our ProActive Protein A coated microspheres and ask about other options 5 We have heard recently about Protein L which is supposed to be a more universal monoclonal antibody binding protein We have not evaluated any yet but it might be easier better than GAM worth consideration For more on Protein L see Tocaj A U Sjobring L Bjorck 0 Holst 1995 High level expression of protein L and immunoglobulin binding protein in E coli J Fermentation and Bioengineering 80 1 1 5 Bangs Laboratories e Ask The Particle Doctor Page 43 Dye Leaching Lateral Flow Tests Cleaning Methods Surfactant Removal Washing Beads Cleaning Methods Contamination Deionized Water Dissolving Beads Divinylbenzene DVB Organic Solvents Toluene Q I am experiencing problems in getting your 0 3 0 5um COOH and amino modified latex beads to chromatograph on paper have conditioned the beads by washing x10 in aqueous buffer to remove surfactants and other small molecules At present am only able to achieve chromatography by using a mixture of aqueous organic mobile phase e g 75 aqueous buffer 25 ethyl acetate Should be using another type of bead for this work or is there another protocol need to adopt for this application Particles probably did not move because 1 they were too large to move through the pores of the strip you chose 2 they were clumped by the buffer and the clumps couldn t
145. otes This complex is not significantly affected by pH values between 2 and 13 nor by concentrations of guanidine HCI up to 8M at neutral pH s This information was originally reported in Avidin Green N M 1975 Advances in Protein Chemistry 29 85 133 1975 New York Academic Press Eds C B Anfinsen J T Edsall F M Richards Q How do make a latex agglutination test for an internal QC check Here is some general information regarding latex agglutination tests 1 LATs make use of microspheres in the range of 0 2 1 0um 2 You can calculate material needs latex antibody and antigen for each test from the following data a it takes 100 latex clumps to judge agglutination b each clump must be 50um in size to be seen by the eye c 10 bonds are required per microsphere to agglutinate them and d sample size can be as small as 10uL See TechNote 301 mmunological Applications for more details When you wantto bind IgG antibody to microspheres there are a variety of options Included are direct adsorption or covalent coupling of the antibody to the microsphere Or you might want to use beads that are precoated with a generic binding protein perhaps streptavidin plus a biotinylated IgG of your choosing A discussion of binding strategies is provided in our TechNote 201 Working with Microspheres TechNotes 101 ProActive Microspheres 204 Adsorption to Microspheres and 205 Covalent Coupling may also help as you cons
146. oval and what you remove may very well be destroyed in the process Bangs Laboratories e Ask The Particle Doctor Page 35 Agglutination DNA Attachment DNA Hybridization Latex Agglutination Tests Ab Ag Binding Reactions Binding Quantities IgG Spacing Latex Agglutination It may be easier to measure protein in solution with BCA or other protein reagent from Pierce or others before and after adsorption on particles Then by difference you will know how much adsorbed You may also use a total protein assay directly with the coated beads Q How can make agglutination tests for DNA with 1um microspheres How fast can one perform such a test A Still trying to sneak in two questions at a time aren t you 1 DNA hybridization based tests and assays can be made which are analogous to antigen antibody tests and assays If the microspheres were coated with a complementary strand of DNA or RNA they could react with the target DNA or RNA in a sample We know of some DNA agglutination work and DNA strip tests BUT that was a big but the method of binding the DNA onto the beads will be critical to your success If you bind by adsorption then the DNA will probably be bound by several or many points of contact to the beads analogous to the adsorption of proteins onto polystyrene Will the target DNA bind to complementary DNA which is bound by several points of attachment Alternatively will the DNA stay bound if you try
147. pensation is correct FITC PE Compensation Standard Trying to sneak in a second question huh We offer two products for color compensation of your cytometer The FITC PE Compensation Standard Catalog Code 820 consists of four different bead Simply Cellular amp populations one labeled with FITC one labeled with PE one labeled with both FITC and PE and an Autofluor Compensation population exhibiting a level of fluorescence similar to that of unstained cells The beads come pre labeled and Standard ready to use The product provides a simple means of setting two color FITC PE compensation The Simply Cellular amp Compensation Standard Catalog Code 550 consists of two populations of microspheres with goat anti Mouse GAM IgG surfaces The two populations have the ability to bind different amounts of mouse monoclonal IgG antibody The user stains these beads with their fluorescently conjugated mouse monoclonal antibody The resulting stained beads exhibit dim and bright levels of fluorescence One drop of beads is stained for each fluorescent antibody which may then be analyzed together on the cytometer Bangs Laboratories e Ask The Particle Doctor Page 21 BioMag BioMag Immobilization Kit immunoassays QuickCal Adsorption BioMag Plus Wheat Germ Agglutinin Carbodiimide Crosslinker Lectin Coating References Wheat Germ Agglutinin To address your second question take a look at the gr
148. perature Next we measured amount of Ab in solution after adsorption and found 0 mg mL for 0 8um particles and 0 3 mg mL for 2 5um particles we tried What happened here You have no problem so far The surface capacity of polystyrene for adsorption of protein is 3mg IgG m The specific surface area of particles in m g is 6 d where d is in um Therefore 0 8um particles will have surface area of 6 0 8 or 7 5m g Thus they will adsorb 7 5 x 3 22 5mg IgG g of particles or 22 5 x 0 05g 1 1mg IgG 0 05g particles You added 1 2mg IgG 4mL x 0 3mg IgG mL to 0 05g of particles or about what we would expect to saturate the surface if it all were to adsorb onto the particles You found Omg of protein mL in the supernatant and this result is consistent with the amount you added and the capacity of the particles If the IgG adsorbed to capacity on the polystyrene surface we would expect to find none left in the solution Likewise the 2 5um particles will have surface area of 6 2 5 2 4m g and will adsorb 2 4 x 3 7 2mg IgG G of particles or 7 2 x 0 059 0 36mg IgG 0 05g particles You added 1 2mg of 19G 0 05 g of particles an amount which is about 3 33X what we would expect to saturate the surface The excess 1 2 0 36 or 0 84mg will be found in the supernatant at a concentration of 0 84 4 0 21 mg mL which is quite close to the 0 3 mg mL concentration which you found Thus we would say that your adsorption results are about
149. phelometric Assays 34 39 Tub ener 30 59 IUrInoP E 42 Two Step Coupling eene 21 42 62 U Ultra Violet B8 citi eere 16 Uniform Magnetic Microspheres sss 24 Uniform Separation sse 12 Urine Based Tests sssseese n 46 UV amp Violet Excitation e 12 16 V ViaCheck Viability Standards 3 Visibly Dyed Beads 45 Ww Washing Beads 16 30 38 43 50 52 53 Water Soluble Carbodiimide ee 59 Water Soluble Polymer WSP sss 38 Wheat Germ Agglutinin seeennne 21 Last Updated March 11 2011 Bangs Laboratories e Ask The Particle Doctor Page 69 Trademarks and Registered Trademarks To the best of our knowledge the trademarks and registered trademarks listed here are accurate as of the printing of this document Bangs Laboratories Inc Absolute Count Standard Autofluor Bangs Laboratories Inc Certified Blankt COMPEL Full Spectrum Latex Course Painless Particles ProActive QC3 QC Windows Quantum QuantumPlex QuickCal Right Reference Standard Simply Cellular Starfire Red SuperAvidin BD Biosciences Cy Chrome GE Healthcare Limited Cy including Cy5 and Cy7 is a trademark of GE Healthcare
150. philic head is presented to the aqueous environment Charged surfactants like SDS are often used which provides charge stabilization in addition to the wetting function There will be residual surfactant from the synthesis itself and additional surfactant would be used to treat sticky aggregated suspensions have tried to conjugate lipopolysaccharide LPS to carboxylated beads with no success Any ideas as to what the problem could be Could you share a proven protocol Lipopolysaccharides often require specialized coating strategies as they typically lack needed reactive groups for coupling at least in their native states Most LPS immobilization schemes feature modification of saccharide moieties for covalent binding There are also chemistries that are more broadly applicable such as the use of epoxide containing reagents oxidation etc though you may want to consider the benefits drawbacks of each see Bioconjugate Techniques ISBN 0 12 342335 X Polysaccharides may also be immobilized via their affinity binding partners lectins however these are reversible interactions If the polysaccharides could be biotinylated they may be for most application conditions permanently immobilized to streptavidin coated beads Another option might be to adsorb LPS to microspheres either through hydrophobic tails of the lipid to non functionalized polystyrene microspheres orthe hydrophilic region to silica microspheres However for ads
151. ptavidin coated microsphere Binding capacities of small microspheres will in general be greater than those of larger microspheres due to increased surface area per unit weight Researchers performing single molecule imaging work DNA have utilized our low binding SA coated microspheres with success better results for their applications than some of the higher binding beads Please note that regardless of binding capacity of the microspheres utilized optimal binding conditions to achieve minimal binding or binding of a single molecule will likely need to be determined empirically Some references regarding use of streptavidin coated microspheres in single molecule imaging AFM studies are available from our literature library at your request Orders for beads may be placed through our Customer Service Department phone fax email info bangslabs com or at our website http www bangslabs com Q Can you please advise how to use our new HIAC Royco instrument to measure your microspheres What s the matter Don t you trust our numbers Previously we used an Accusizer 770 Particle Sizing Sytems which depends upon the same methodology i e photozone method as the HIAC Royco It was at its best with very dilute samples For the Accusizer 770 we used 100 500 microspheres mL Note We used much more dilute suspensions than the manufacturer recommends The use of dilute suspensions lowers coincidence multiple particles in the ch
152. r methacrylic acid with or without surfactant etc During early experimentation feasibility you might try beads made by different processes manufacturers until you find the combination of bead and process which yields the desired result After you optimize the binding of your protein to one microsphere lot then when you try to make a replicate binding lot make sure you are really using a replicate bead lot Bangs Laboratories e Ask The Particle Doctor Page 40 Catalog Code Meanings OEM Catalog Numbers Blocker Concentration BSA Sonication Hydrophilic Beads Large Microspheres Nitric Acid Sulfuric Acid 5 When testing your process for robustness Will your process work if you change bead source or manufacturing process you again might want to try beads made by different processes 6 Talk to us and tell us what you are trying to do at all stages of your work If we know you are in early feasibility testing we can point you at beads from different processes When we know that you are trying for replication we ll be sure you get replicate bead lots even if we must custom produce another lot made for you Q Please help me understand your catalog codes and lot numbers How do I choose the microspheres need Our Certificates of Analysis COAs have two product identification numbers on them The first number specified is the catalog code e g DS02B This is a generic code that applies to all plain polystyre
153. rage you to put them to work in your application we think you ll like them saw your recent ad announcing your new anti human beads Sounds like a top secret government conspiracy to me What s the deal SEDAN ii y Are you related to that guy who always writes in every time we mention goat j i anti mouse and asks about antimurine bias Well don t worry there s no evil plot here The beads you re referring to are the new Quantum Simply Cellular Human Antibody Binding Standards The anti human nickname comes from the Goat anti Human antibody found on the beads surfaces The kits allows you to measure Bangs Laboratories e Ask The Particle Doctor Page 25 Freezing Prevention Shipping Methods Blocker Concentration Buffers Conformational Changes IgG Coating Levels Reagent Stability References the Antibody Binding Capacity ABC of cells when you re using a fluorescently labeled human antibody as your reporter Here s how it works The kit consists of five bead populations one blank population and four populations labeled with varying amounts of Goat anti Human IgG Each of the four labeled populations is calibrated to bind a specific number of Human IgG class I or II monoclonal antibodies calibrated to ABC values These beads are stained with your human monoclonal antibody just like you stain your cells When you run them on the cytometer you ll get five separate peaks of fluores
154. re are a few options If for example you have a mouse mAb that is labeled with your fluorochrome you could use it to stain protein A or anti mouse IgG microspheres to create your own fluorescent standard Be sure to check the Ab subclass if using protein A We have a number of options in 5 10um polymer beads that would be suitable for flow See our online Polymer amp Silica Beads catalog for details We also sell a wide range of fluorescent bead standards that may serve as reasonable surrogates if you simply wish to check the laser or detector for your fluorochrome Our flow cytometry Reference Standards span the spectrum from UV to Far Red Additionally we have several internally labeled fluorescent microspheres in our standard catalog Reference spectra for internally labeled spheres are now available in the Technical Literature section of our website TechNotes 103 and 103A I m interested in using PolyLink EDAC coupling to attach antibodies to COOH functionalized beads However this method seems to attach to any available amine group of the protein so I m not sure if should use it Oh it ll be fine go ahead and use it How we do love to sell beads and reagents OK OK On a more serious note using carboxylated beads with EDAC will result in some level of nonspecific orientation although it is generally accepted that the Fc region will preferentially orient toward the bead based on antibody packing and the slightly grea
155. roduced a quantitative EGFP microsphere standard for BD Clontech this was discontinued after 2004 due to licensing The development of GFP microsphere calibration standards is covered by US Patent 76 326 157 Recombinant fluorescent protein microsphere calibration standard Q After oligo coupling how can determine the efficiency of the oligo immobilization There are a number of methods for assessing binding efficiency 1 Binding capacity of oligo dT beads may be determined by binding mRNA and measuring the amount of eluted RNA by wavelength scanning see our TechNote 302 2 The amount of bound oligonucleotide may also be estimated by hybridizing it to its biotinylated complement with detection via a streptavidin labeled fluorophore A similar approach is detailed in Kumar A et al 2000 Nucleic Acids Res 28 14 e71 Available for free download from the journal website 3 Use radiolabeled probes Lund V et al 1988 Nucleic Acids Res 16 22 10861 10880 Day P J R etal 1991 Biochem J 278 735 740 4 Nucleic acid dyes stains may also be used to determine the amount of DNA attached to beads or unbound in solution Walsh M K etal 2001 Bangs Laboratories e Ask The Particle Doctor Page 27 Carbodiimide Reaction CML Binding EDAC Ethanolamine Quenching Surfactant Two Step Coupling Biotin Elution Competitive Detachment References Streptavidin Coated Microspheres Photobleaching Quenching
156. rs populations may be stained and run separately for optimal gating Broad peaks may indicate that saturation has not been achieved an antibody titration will aid in ensuring that bead samples are stained to saturation Ensure that only singlets are gated Do not stain the blank population which consists of uncoated polymer beads that will be happy to bind antibody nonspecifically QuickCal f the curve doesn t fit in the window it s likely that the wrong version of the template has been used To determine resolution or the appropriate version of the template look at the x axis of the fluorescence histogram Typically numbering of 0 1000 1024 template 10 10 BD Relative Linear 10 10 Coulter Relative Linear An unexpectedly high detection threshold may indicate free dye in the system or that the blank bead population was stained with the antibody coated beads If you achieve poor results with a particular run stain and run a new sample Staining and running peaks separately may provide more specific information for troubleshooting Labeling beads with a different antibody clone and fluorophore will aid in identifying clone or fluorochrome specific effects And of course we re happy to offer additional comments support or a warm shoulder if needed Q I m having problems when centrifuging microspheres My bead counts are so low after staining and washing that I m afraid they re being broken Is this pos
157. rvous about performing these analyses Can you offer any tips beyond the standard protocol Any common pitfalls should avoid First let us say Welcome to Flow Cytometry We like it and we re sure that if not immediately then in time you will too If your institution has a core flow cytometry facility you re in luck as you ll have access to experts in sample handling instrument operation etc Quantum Simply Cellular amp products are somewhat specialized and do presume a basic proficiency in these techniques much of which will translate to the use of antibody capture beads However if you re own your own and even if you re not we wouldn t think of abandoning you We are happy to provide you with additional suggestions up front as well as troubleshooting tips in case things don t go quite as planned Getting started Conduct an antibody titration for the beads so that you re confident that saturation is being achieved Bear in mind that the antibody concentration used for cells may not be optimal for the beads e Stain and run each antibody coated population Beads 1 4 separately for at least the first run to ensure satisfactory labeling and optimal resolution for gating Use the same lot of the same Ab clone for the duration of the study Where a new lot must be used run bead samples stained with each lot in parallel to identify Bangs Laboratories e Ask The Particle Doctor Page 8 Centrifugation any vari
158. s may behave very differently during adsorption One major difference is that they often have very different isoelectric points Pc IgG adsorbs best at its isoelectric point pH 7 8 At this pH one gets maximum adsorption apparently because the IgG is in its most relaxed state and each molecule takes up lowest area on the surface Do you know the isoelectric point of your Mc Ab Some Mc Ab s have isoelectric points as low as pH 4 Adsorption under these conditions may work for you At these low pH s some PS polystyrene particles may not be as colloidally stable they may tend to spontaneously clump or flocculate so other tricks may be necessary to get good adsorption One customer used ascites fluids for coating the particles he got good coating and activity but immunological stability was poor perhaps due to the enzymes which denatured the protein after a few days or weeks depending upon temperature This customer is now trying to partially purify his ascites fluid to remove the problem It may be better to start with purified Mc Ab and to add things like coadsorbents or blocking agents It is well known that pure surfactants do not adsorb as well as those which have an impurity which is also surface active the two adsorb better than either alone So try mixing Mc Ab with BSA HSA non ionic surfactant or other blocker It is quite reasonable to expect that some other protein will make a more compatible environment for the Mc Ab A rece
159. s the surfactant diffuses out of the solution and off the microspheres the protein will replace it on the microsphere surfaces This dialysis method is still quite passive you must wait for the surfactant to diffuse through the membrane Now ultrafiltration membranes are available from Microgon for example with 50 000 MW cut offs In hollow fiber form these membranes can be used to hold protein and Bangs Laboratories e Ask The Particle Doctor Page 51 DNA Adsorption Protein Adsorption Silica Microspheres Lateral Flow Tests Nitrocellulose Membranes Strip Tests Equations Surface Area Equations Parking Area Stokes Diameter microsphere together while surfactant is actively under pressure washed away and forced out of the coating zone After such surfactant cleaning the microspheres should be quite well coated with protein Then transfer the microsphere protein suspension to a 0 1 or 0 2um membrane cross flow filter and remove the unbound protein from the microsphere suspension Q How do I adsorb protein onto your silica microspheres A The short answer is You don t The long answer is that there is good news and bad news First the bad news Remember that protein adsorbs onto polystyrene latex very well because IgG forms many hydrophobic bonds to the PS surface These bonds hold the protein onto the surface indefinitely and almost irreversibly Just try to get it off On the other
160. sible Polystyrene based microspheres can handle the rigors of centrifugation and we expect this separation technique to work well for spheres gt 0 5um When you hear us cautioning against overzealous centrifugation we re most concerned about having too tight of a pellet form i e irreversible aggregation of microspheres If spheres are disappearing it s most likely that they are becoming more hydrophobic with successive washes as the surfactant concentration is lowered and are clinging to the sides of tubes You may centrifuge them with a bit more force to sediment them or add back a bit of surfactant to aid in wetting Bangs Laboratories e Ask The Particle Doctor Page 9 Long Term Storage Polystyrene Microspheres Stability Calibration Standards NIST Traceable Particles Particle Sizing Some general guidelines fora benchtop microcentrifuge follow 7 3cm rotation radius 5 minute centrifugation These may be used as a starting point for further optimization if needed Uncoated Polymer Protein Coated Polymer 0 5um 6 500 14 000 x G gt 0 5yum 8 000 11 000 x G 1 0um 3 000 5 500 x G 1 0um 5 500 8 000 x G lt 5 0um 1 300 3 000 x G lt 5 0pm 2 000 5 500x G Silica gt 0 5um 3 000 5 500 x G 1 0um 1 300 3 000 x G lt 5 0um 750 1 300 x G Additionally to better understand the efficiency of centrifugation steps you might examine samples of the supernatants under the microscope 40
161. sorption PNAS 97 16 9037 9041 Szleifer J Satulovsky 1999 Kinetic and thermodynamic control of protein adsorption by grafted polymer layers Polymer Preprints America 40 2 89 90 Some of their work pertains directly to the attachment of PEG to surfaces to prevent protein adsorption if these articles are not of direct relevance to your work their references sections may prove to be helpful Inthe process of cleaning microspheres it seems likely that you will change the solids concentration and lose a fair fraction of particles especially with small amounts of latex How do we determine the solids concentration after washing First we agree that it js important to know the amount of particles you have at any stage of your process to know the weight of particles per mL so they can be handled conveniently and to control your process for addition of the proper amount of wash solution and coating materials Second you will lose some particles in washing more or less depending on which kind of particles you have Less if you are working with monodispersed size microspheres which should all behave the same While there will always be some losses in transferring things there won t be too many losses unless the particles are caught in filters More will be lost if you are working with smaller samples and smaller particles harder to handle Losses will also depend on your cleaning process Of course we won t mind if you wan
162. sts Sandwich Strip Test Strip Tests Aggregation Electrophoretic Mobility Instruments Field Flow Fractionation Latex Immunoassay Monodispersity Nephelometers Particle Counters Particle Sizers References Spectrophotometer Latex Agglutination Tests Particle Determination How do we make a test using dyed particles which move along a strip and form a colored line if antigen is present in a sample Usea pregnancy test as an example In this test Ab coated blue dyed particles flow along a nitrocellulose strip to reach an immobilized second Ab if HCG is present a sandwich forms and results in a blue line for a positive test For this type of test one needs darkly dyed particles which will move easily through the strip The particles should be large enough to give a strong signal as large a light bulb possible yet small enough to move freely along the strip A compromise is made between large bright particles and small fast moving particles See TechNote 303 Lateral Flow Tests Here s the commercial message We have a virtual rainbow of colored and fluorescent particles in many sizes and with different surface chemistries We are currently assessing the potential of latex particles as a solid phase in immunoassays During coating or storage the particles may aggregate We are interested in methods for the determination of the proportion of single particles doublets triplets etc The detection of partic
163. survive centrifugation better than polystyrene particles PS particles are more likely to become firmly stuck together and therefore hard to redisperse And the cleaner they get the more resistant they are to redispersion If you are centrifuging particles to wash or clean them then there are other better and easier ways to clean them Ask for our TechNote 203 for an exhaustive or exhausting discussion of cleaning methods One of these better ways is cross flow filtration which cleans the particles without clumping them Gradually the folks at Spectrum Labs are winning over the particle world converting the biggest particle users to their system Contact them for more information at www spectrapor com Are you still interested in centrifuging particles If you are that persistent then please consider continuous centrifuging In a continuous centrifuge you never need to form a filter cake which is where the particles become clumped Rather you separate the input stream into two fractions and form two output streams a particle rich concentrate and a particle free supernatant discarding the supernatant Then you can dilute the concentrated particle suspension with water or buffer and put in through the continuous centrifuge again until you achieve the appropriate level of cleanliness Q How can you help us to make a new diagnostic test A Thanks for asking We can help in several ways as follows 1 2 3 4 Give us a cha
164. t magnetic separations even from difficult e g highly viscous samples BioMagPlus particles undergo additional processing for removal of fines Composition Silanized iron oxide Morphology Irregular Cluster Surface groups COOH and NH available Density g cm 22 5 Iron oxide content 96 gt 90 Magnetization emu g 25 35 Surface area m g gt 100 Particles g 1x108 Once you ve determined which type of bead to use consideration moves to the surface that will be most effective Often separation is performed using some sort of affinity system for example antibody antigen interaction or charge mediated purification Protein coated magnetic particles are available off the shelf with streptavidin protein A secondary antibody primary antibody oligo dT 20 or anti CD marker surfaces Functionalized particles are used in situations requiring the attachment of less common ligands Q Do you have any products that support quantitative applications in molecular biology Our Quantum MESF kits have been utilized to quantitate fluorescence intensity for Flow FISH Fluorescence in situ hybridization and bead based hybridization assays via flow cytometry Provided that you re using a fluorescent reporter for which we offer an MESF kit FITC PE PE Cy 5 APC and you re evaluating the fluorescence of a particle beitafluorescently labeled cell or microsphere our kits permit quantitation of fluorescence intensity in M
165. t to buy more microspheres to ensure that you will have enough The only reliable way that we know of to measure solids content is by loss on drying We have found good reproducibility and agreement with others measurements by using as little as 100uL of a well dispersed completely resuspended as single particles suspension at 10 solids 100 mL at 10 solids 10 mg which requires a pretty good balance If solids content is 1 then it will certainly take more sample to get good measurable solids This method may be poorer for low solids but it works and it s our method of choice Automated particle counters are also useful for determining concentrations We shouldn t mention any bad ideas but occasionally somebody tries to use a spectrophotometer to measure solids content by measuring the turbidity of particle dispersions This is a bad idea because turbidity depends on particle size and degree of dispersion as well as on particle concentration Thus the absorbance of a 1 solids dispersion of single microspheres will be significantly different than 196 solids suspension of doublets And 1 of 0 2um microspheres will be very different from 1 of 0 8um microspheres Also everything else in the aqueous phase and on the beads will influence the absorbance This is a really dangerous method like skiing out of bounds you are really in avalanche territory It might be possible to devise a method whereby microsphere solids and water are d
166. tein such as BSA to block as well am particularly concerned about the effect that a protein will have on access to the small molecule now linked to the surface of the bead I would like to covalently attach a small molecule to your 32nm COOH modified beads and use this ligand to capture receptor proteins Here s my plan Clean the beads by passage through a PD 10 column or use some form of dialysis then re concentrate beads to 1096 solids Can make single dry beads here Activate the beads with WSC and NHS or HOBt for 3 4 hours at 22 C Dilute the reaction with 0 05 Tween 20 in 200 mM K HPO pH 8 0 which contains the small molecule at 1 5 mM at 22 C then quench residual active esters after 20 hours with 5 mM ethanol amine Bangs Laboratories e Ask The Particle Doctor Page 42 Two Step Coupling Adsorption Antibody Orientation Covalent Coupling ProActive Protein A Microspheres Protein L References Secondary Antibody Binding A The PD 10 column seems like a good approach Normal dialysis tubing of the Pierce Slide A Lyzer should work as well Prewash any column or dialysis tubing well to remove whatever was used to make it Cross flow filtration from Microgon Millipore Filtron Pall Amicon A G Technologies et al can be used to concentrate up to 10 solids or more The Microgon system at least can be used to clean and concentrate the beads Some folks have reported cleaning reacting remov
167. ter hydrophobicity of this domain This immobilization strategy EDAC activation of bead COOH groups generally results in sufficient antibody bound with good activity and is one of the most common for standard applications such as immunoassays We typically recommend directed binding for special cases e g if the Ab isn t performing when traditional covalent immobilization is used or for certain ligands such as oligonucleotides peptides hormones etc If you decide to explore directed immobilization strategies to orient the antibody you might consider use of an Fc binding protein such as protein A or G although you ll need to confirm the protein s Ab affinity these proteins do exhibit variable binding depending on the species in which the Ab was raised as well as Ab subclass Use of an Fc specific antibody would also be appropriate You could also digest the antibody for immobilization of F ab fragments or oxidize the carbohydrate in the Fc region to create aldehydes for direct covalent immobilization or use of a targeted biotinylated linker and a streptavidin support These methods are certainly valid but they do involve additional steps So Go PolyLink Q want to purify my target cells from a fairly nasty sample matrix Ultra high purity isn t necessary but I want to capture as much target as possible Which type of magnetic bead should use A Magnetic particle selection is often driven by practical matters such as ava
168. th ethanolamine Avoid a urine centrifuging step to make your test more marketable You could try filtration of the urine samples to remove the protein Check out Sokoloff R L J M Reno August 20 1985 Method for reducing nonspecific interferences in agglutination immunoassays by adding a halogen substituted carboxylic acid U S Patent 44 536 478 To avoid clumping during 2 step covalent coupling of guinea pig polyclonal antibody to carboxylated beads I added 0 05 Tween 20 just before EDAC carbodiimide addition The Tween inhibited the clumping induced by EDAC addition but the agglutination between the bead and antigen showed poor antibody coupling on bead surface Do you have a solution to my problem Certainly If too much Tween is added it could interfere with coupling by coating antibody or beads to prevent their coming together Add only enough Tween to prevent clumping maybe 0 005 Tween while permitting good coupling Test this by adding serial dilutions of Tween to microspheres and mix with EDAC buffer in the absence of antibody Then when you get Tween dilution which is just enough to yield a stable microsphere dispersion in buffer test for adequate covalent binding After I bind a protein onto my microspheres they will be stored in a Tween 20 containing buffer Do you expect that the protein will be desorbed under these conditions Is it possibly better to use covalently binding beads Yes and Yes A small
169. thods one for surfactant removal before protein coating and one for excess protein removal after coating Practical Answer Dr Seaman says that water is free of surfactant when the foam or bubbles on top of 5mL H O shaken in a 10mL test tube collapse in 2 3 seconds You can use this indicator to test supernatant coming from the microsphere clean up whether by centrifuge or filter system We call this the Seaman Shake Test For full details see TechNote 203 Washing Microspheres How do we know that we have not contaminated the microspheres in the cleaning process Possible Answer Make sure that water going in is as clean as you can get no ions no organics no microbes Otherwise microspheres can pick up stuff from the water Our deionized DI water system has a mixed ion exchange bed with a conductivity meter organic filter recirculating filter system with UV light to kill bugs and a 0 2um final filter It s the best water we know how to make or get and we use it directly from the system no stored water for things to grow in Monitor things with a microscope to ensure that you always have singly dispersed microspheres What kind of organic solvents can one use to disperse and dissolve your microspheres and how do I get them out of water and into the solvent Most of our microspheres are made of polystyrene PS or copolymers of styrene and divinyl benzene S DVB The DVB crosslinks the polymer chains and provides stre
170. tion of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library Nature Biotechnology 21 163 170 McConnell S J et al 1999 Biopanning phage display libraries using magnetic beads vs polystyrene plates Biotechniques 26 2 208 210 214 Yeung Y A K D Wittrup 2002 Quantitative screening of yeast surface displayed polypeptide libraries by magnetic bead capture Biotechnol Prog 18 2 212 220 We offer BioMag COMPEL and ProMag microparticles with a number of coatings for affinity binding e g secondary antibody streptavidin protein A etc in addition to BioMag and COMPEL functionalized microparticles for covalent attachment of capture molecules See our Magnetic Particles brochure and TechNote 102 for information on particle characteristics I would like to couple antibody to microspheres but I haven t worked with beads before What do you suggest Using our custom coating service naturally Seriously although we do offer a custom coating service we do have products that are intended to introduce users to the wonderful world of microsphere coating in a relatively painless manner Our new PolyLink Protein Coupling Kitincludes EDAC for activation of COOH surface groups on microspheres coupling buffer and wash storage buffer The accompanying protocol Product Data Sheet PDS 644 has been optimized for use with polymer microspheres 1um and larger We also offer coupling kits fea
171. tions Crosslinking Protein A Microspheres Flow Cytometry Quantum Simply Cellular amp Surface Marker Expression Troubleshooting Tips Well first you must stand on one foot Sigh The jokes just don t seem to get any better do they As a general recommendation you should rigorously standardize all aspects of the coupling process both the written protocol and its execution Though this seems obvious it s important to take a critical look at your process including washes reagent addition incubation and mixing steps etc and raw materials reagents base beads buffers etc Seemingly small deviations or inconsistencies can show themselves through variability in your results If you re confident that reagents and protocols have been consistent would suggest examining the EDAC activator which should have the appearance of a free flowing white powder EDAC is extremely hygroscopic and will absorb water with disastrous results Persistent clumps are evidence that the reagent has been contaminated with moisture and it should be discarded and fresh EDAC obtained New vials of EDAC should be stored desiccated at 20 C and warmed to room temperature in a desiccator before opening to avoid condensation If possible the headspace may be flooded with argon before the vial is re sealed and stored If you haven t settled on a specific protocol you could use our PolyLink Protein Coupling Kit Catalog PLO1N as a new
172. to hybridize while it is on the beads It may be necessary to bind to the beads by covalent attachment by one end of the complementary DNA molecule so it is free to hybridize properly See TechNotes 205 and 302 for more information 2 The old latex agglutination tests LATs using 0 3 0 8um particles or microspheres at 1 solids on glass or paper slides take about 2 3 minutes with active rocking or rotating With 1pm particles it should be about the same time For reverse passive agglutination RPLA tests done in 96 well plates like hemagglutination tests it may take 1 hour or longer depending on particle and liquid density since the particles must settle to the bottom of the wells and form a lacy pattern or button Of course those estimates were for protein based immunoassays For DNA RNA based assays more time may be required for the strand hybridization reaction to occur See our TechNotes 201 and 301 for more answers about LATs have successfully coated some plain PS beads with polyclonal rabbit IgG using your advice coated 250uL of 10 250nm beads with 0 8mg of IgG I added 2mg but only 0 8 bound 1 2 the capacity of the beads by my calculations My assay is in glycine buffered saline pH 8 2 the same buffer used to coat the latex The total assay volume is 900LL including the 300uL of antigen at concentrations 2 4000 ug L tried 0 1 0 5 latex solids in the assay I can detect no antigen dependent change in
173. trated by evaporation after repeated bottle openings and a possibly loose cap sometime Higher solids requires more Ab mL to coat And if evaporation occurred then the increased concentration of surfactant could also interfere with the adsorption of protein and yield a less stable product 3 Although not related to your problem microbial contamination can result from continual reopening of a bottle of beads Azide or merthiolate will prevent this We believe that the particles are stable for years or even decades unless contaminated Surfactants are chemically stable too Thus we encourage customers to add antimicrobial to the beads and to repackage them into appropriately sized aliquots as soon as they receive them We can also prepare aliquots of any size and add your favorite antimicrobial when we prepare your order if you wish Hint One of our customers has their beads packaged in aliquots which exactly fit their production recipe So no need to measure beads during production They just pour it in Desorption I have coated protein on some 0 2um dyed beads by simple absorption I would like to know if there is any way to desorb the protein from the beads so that a Protein Quantification quantification of these proteins is possible Removing protein from PS beads is about as much fun as getting chewing gum out of your sister s hair There are recipes using high surfactant temperature and pH but you are likely to get incomplete rem
174. turing BioMag Plus Carboxyl see PDS 618 or Amine see PDS 617 particles and aBioMag Magnetic Immobilization Starter Kit that includes a magnetic separator reaction flask particles and buffers see PDS 546 Our TechNotes 201 206 provide additional support on a range of topics related to microparticle handling Simply select the microspheres and or kit that best meet your needs review the coupling protocol and pertinent TechNotes and you ll be off and running Or heck join us at The Latex Course In our flow lab we run calibration beads daily to verify instrument sensitivity and adjust detector settings I m wondering if we should also periodically run reference beads as a check on optical alignment and which ones would be best Our Full Spectrum microspheres are internally labeled with multiple fluorophores to excite from the UV to the near IR and emit over the full range This product is suitable for instruments with single or multiple lasers and may be Bangs Laboratories e Ask The Particle Doctor Page 19 Cell Quantification Flow Cytometry Absolute Count Standard Immunophenotyping Quality Control Quantum MESF Kit Simply Cellular used to check the alignment of all of the lasers and PMTs Histogram showing fluorescence and narrow CV s of the Full Spectrum Catalog Code 885 standard need to quantify the number of white cells in a sample for a study am doing My study is
175. what we would expect For more information check out TechNote 204 Adsorption to Microspheres After adsorption of Ab we washed and resuspended with BSA buffer We next tried agglutination of particles using a virus culture but no agglutination appeared In this case agglutination should be caused by a virus attaching itself to two or more antibody coated particles and binding the particles together You might need to adjust the amount of virus added to cause agglutination If your sample contained a very high concentration of virus they could completely cover the antibody coated particles and no agglutination could occur because no bridging between particles could happen Try diluting your virus concentration by 10X and 100X to see if you can get agglutination this way In some cases you may also need to adjust the amount of antibody on the particle surfaces to less than a monomolecular layer See our TechNote 301 regarding coating density of Ab or Ag on the particles and the ratio of agglutinating agent to particles Q How do I bind DNA or oligonucleotides on your particles You can use magnetic particles amino or COOH modified silica or polystyrene based particles There are several binding methods depending on the molecular weight of the DNA or piece you use and on the type of particles you choose 1 DNA will adsorb directly onto our uniform silica particles if the conditions are right Silica will be negatively charged
176. y Turbidity is sensitive to particle composition refractive index diameter solids content and wavelength of light used If composition diameter and wavelength are held constant i e for the same lot of particles measured under the same light conditions then it is possible to measure and plot turbidity absorbance in a spectrophotometer vs concentration Such a curve would be most sensitive in very dilute ppm concentrations The accuracy of the data would be very sensitive to the care involved in doing the work especially accuracy of dilution taking 10 solids suspension and diluting it carefully to the ppm range thorough redispersion no particles sticking together and absorbance of the diluent water surfactant for dispersing particles other ingredients A wavelength of 700nm is suggested Obviously such a curve would be applicable only for particles of the same lot measured under identical conditions spectrophotometer wavelength diluent etc and a new curve would be needed for each different lot of particles If particles are coated with protein or other chemicals then a new curve should be generated Bangs Laboratories e Ask The Particle Doctor Page 60 Cell Depletion Magnetic Particles Phagocytosis Filter Method Fluorescent Beads Immunoassays Quenching References Magnet Selections Rare Earth Magnets Adsorption Blockers Covalent Coupling Hydrophilic Beads Nonspecific Binding D
177. y also be helpful Q What are these strip tests you refer to Strip tests are like sandwich assays but the color comes from dyed particles which complete the sandwich instead of an enzyme and substrate to generate color see TechNote 303 Q What s the best way to bind protein to particles and avoid NSB Answer 1 Adsorbing protein onto polystyrene and blocking with BSA gelatin or other protein works well But we cannot guarantee that some other protein won t adsorb also Answer 2 Covalent coupling of protein to carboxylate modified particles permits permanent anchoring of protein to the surface This may be followed by addition of large amounts 1 596 of surfactant like Tween 20 with or without blocker protein The amount of surfactant or blocker can be increased until complete blocking is achieved without worry that the desired protein will come off Can I activate COOH modified particles with WSC water soluble carbodiimide today and couple my protein to the surface tomorrow We recommend adding protein to the activated particles immediately after activation to prevent hydrolysis of the activated beads Q How do measure solids content especially for dilute lt 1 particle suspensions The best way we know to measure solids content is to weigh suspended beads evaporate the water and weigh the solids remaining There is no simple instrumental method to give solids or number concentration of particles with accurac
178. y test researchers had trouble putting reproducible spots of antibody on the membranes They finally decided to coat particles with protein in a flask controllable homogeneous chemistry possible and spot the particles onto the membranes easier to control where the particles go both in depth and width We can help with your choice of microspheres to use and how to coat them Q What is the surface area of the microsphere and how many microspheres are there per gram or per mL See our TechNote 206 for the equations and sample values For polystyrene p 1 05 g mL For silica p 2 0 g mL We report all calculated N s particles per g and per mL and A s on Certificates of Analysis or you can ask for these values anytime Of course if you have any questions just call fax or e mail us Snail mail is OK too In June Deep Coat reported that you could put 75 000 IgG s on one 0 8um microsphere Do you really believe that A That data came from Dr Singer so you can ask him about it When we calculate it using 10nm for the Stokes diameter of IgG we get 25 600 IgG s 0 8um Bangs Laboratories e Ask The Particle Doctor Page 52 Surface Area Blockers Clumping Coating Bead Concentrations Coating Beads Cross Flow Filtration Washing Beads Centrifugation Continuous Centrifugation Cross Flow Filtration Equations G Forces Particle Density The close packed parking area for a spherical IgG molecule a
179. ytometers Initial Target Channels ac3 QC Windows Quality Control Anti CD Markers BioMag Cell Separations Q What type of daily QC do you recommend for flow cytometers For typical QC needs we recommend QC Windows which is the product that we use for daily set up and QC The QC Windows kit consists of QC3 and Certified Blank microbeads It is supplied with Initial Target Channels that when used in conjunction with labeled control cells provide a unique approach to unified instrument setup and qualitative evaluation of instrument performance QC Windows allows multiple users to establish a Common Window of Analysis with respect to the fluorescence intensity Instruments that have been adjusted to a Common Window of Analysis produce histograms that are nearly identical Such standardization may be critical when comparing the presence absence or relative intensity of immunophenotyping cell clusters in bone marrow or leukemia samples It also allows data comparison independent of instrument make QC Windows is an essential part of a uniform set up protocol to establish a Common Window of Analysis The QC3 standard has the same spectral properties and fluorescence intensity as the samples being analyzed QC Windows establishes the position of the F NUMEN mum fluorescence intensity Window of Analysis E Sae SANIP O 1458100 LOCUM ASTU EE OT y y Window of Analysis was established using QC Windows on each instrument b

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