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1. read that only proteins from 20 kDa to 200 kDa can be labeled with your ChromaLink kit Our protein has a MW of 5 kDa Does that mean that the kit will not work for us While our ChromaLink reagent is certainly compatible with small proteins or biomolecules our kit is comes with several desalting apparatuses that have a protein cut off of 7 000 Da 7 kDa We could certainly modify our kit for you and add some diafiltration units with a cut off of 3 kDa We would only recommend the diafiltration units if you have significant amount of your small protein we are a bit concerned about the protein crashing out on the membrane If you have an abundance of protein we would certainly recommend trying the kit with this diafiltration procedure Also the recommended amount of ChromaLink added to your small protein would of course have to be adjusted Is your ChromaLink biotin labeling kit useful to biotinylate DNA oligonucleotides Our labeling reagent reacts with a primary amine group on any biomolecule Therefore in order to biotinylate your oligo you would have to have it synthesized with an amino modification on either the 3 or 5 end Most oligo companies can directly add a biotin onto the nucleotide as easily as they can add a primary amine group The ChromaLink reagent would therefore require an extra step and while it would be slightly more quantifiable you could determine EXACTLY how many biotins oligo a mass spec from the ol2. Add the aliquot directly into a previously prepared solution of 1 M NaCl containing 1 Tween 20 and gently pipette up and down several times to disperse the beads while avoiding foam 3 Immerse the tube containing the beads into a water bath sonicator e g Branson 1200 120 volts 50 60 Hz for three min 4 Remove a small aliquot of the beads and place them on a standard light microscope e g 100X to 400X to confirm monodispersity 5 Once monodisperse the beads can be washed twice with a magnet using molecular grade water Note once the beads are monodisperse they take a lot longer to pull them to the walls of the tube using a magnetic field up to two full min 6 Once the beads are monodisperse they can be placed into any other suitable buffer you wish with or without Tween 20 and the majority of the population will remain monodisperse Over time for example overnight the beads will begin to reform aggregates due to the nature of streptavidin itself This cannot be helped Important comments and notes 1 M NaCl containing 1 Tween 20 combined with sonication does not adversely affect the biotin binding capacity of the NanoLink beads Do not attempt to sonicate or disperse larger masses of beads at one time or in a larger volume Attempts to disperse the entire volume of beads or even a smaller volume such as 100 uL beads 1 mg at a single time is not effective A large amount of sonication energy must be focused on a small
3. 1 conjugates We suggest modifying the 30 kDa protein with HyNic and the antibody with 4FB For HyNic use 15 20X mole equivalents to have 2 3 per protein For 4FB use 10 20X mole equivalents to have 3 5 per antibody Make a 1 mg mL solution of each for modification After a 2 hr modification pass 130 L corresponding to 130 pg over the desalting column should get about 100 ug of modified protein With these 2 tubes make some combinations for your conjugates incubating 2 hr with TurboLink buffer Cat No S 2006 105 and run the products on a gel to see which ratios give the best 1 1 conjugate Scale up your conjugations after getting results from the gel Gel lanes Protein alone Antibody alone Pair wise combinations can set this up using both modification conditions for both your antibody and protein 0 5 1 1 1 1 0 5 For quenching the conjugation reaction add a 50 fold molar excess of sulfobenzaldehyde to the reaction to react with any excess HyNic groups How long are the linkers stable on my modified biomolecules AFB linkers are extremely stable on the activated biomolecule and have remained 100 active for over a year when stored at 4 C The HyNic modified biomolecules should be reacted to their 4FB counterpart within 3 hours after modification Does Solulink have any PEGylated linkers suitable for appending to gold surfaces At the present time Solulink has no products that can attach to gold particles Do you offer
4. HyNic and 4FB m trying to crosslink peptide 3 000 KDa with protein 50 000 KDa To modify theses use S HyNic for the protein and S 4FB for peptide When I mix the peptide with S 4FB 1 pg of S4FB 20 pl of DMSO I always see turbidity and a precipitate Do you know why this happens Peptides can be particularly difficult to modify due to their propensity for low aqueous solubility Depending on the sequence the peptide may only be soluble at high or low pH due to acidic or basic residues Additionally some residues such as A F L V are very hydrophobic at any pH Since the S 4FB will modify all lysines present as well as the N terminus it may be possible that the peptide is being over modified with 4FB Thermo Scientific offers some tips for working with troublesome sequences Ultimately it may be necessary to either optimize the sequence by substituting hydrophilic residues or by putting the HyNic on the peptide during SPPS You can also change the strategy by putting the more hydrophilic HyNic on the peptide and the 4FB on the protein but beware it is easy to over modify proteins with 4FB and they too will precipitate would shoot for 2 3 4FB molecules per mol of 50 KDa protein ie add 5 equivalents to the crude reaction with the protein at 3 mg mL How stable is my 4FB labeled oligo After modification of your 4FB labeled oligo you can store it at 20 C in ANY buffer will be very stable Lyophilized is best but
5. MW greater than 7 000 It is possible that you experienced some precipitation of your protein immediately after modification If the protein concentration is too high for some antibodies aggregates can form and a precipitate forms If this is the case you might try lowering the Ab concentration to around 1 mg mL With some antibodies lysine modification removal of charged groups can drastically affect solubility If this is the case you might try modifying with a lower MSR Biotinylate your antibody again using a lower antibody concentration solulink How do I store the biotinylated IgG control Once resuspended in buffer you can store the biotinylated IgG control at 4 C and it should be good for 1 2 weeks Does ChromaLink Biotin bind to metal resins The ChromaLink biotin WILL bind to the metal resin We have observed this before with some of our studies The antibody that purchased is mixed with a protein stabilizer gelatin Can proceed with the labeling reaction anyway without getting rid of the gelatin Most of the antibody would still be labeled correct The gelatin will compete with the antibody with regards to biotin labeling It is strongly recommended that the gelatin be removed prior to labeling The kit will not work as well with the gelatin present If the gelatin or BSA is not removed a mixture of labeled antibodies and proteins results There are antibody purification columns available from other vendors
6. any amine to sulfhydryl crosslinkers with polyethylene glycol PEG spacer arms solulink We have 2 crosslinkers that can react with thiol groups MHPH Cat No S 1009 which is maleimido HyNic and MTFB Cat No S 1035 which is maleimido 4FB Our unique system is that HyNic reacts with 4FB to form a stable hydrazone bond We have a long chain crosslinker PEG4 PFB Cat No S 1034 which reacts with amine groups and attaches 4FB Once our HyNic TAT is in conjugation buffer should it be used within 1 2 weeks What is the stability Once in solution the peptide should either be used ASAP or frozen at 80 C Be sure to aliquot the peptide solution into usable portions prior to freezing to avoid the need to freeze thaw We have found HyNic peptides in water to be stable at 4 C for months We are using your protein oligo kit to attach peptides to oligos My peptide precipitates out of solution once labeled with HyNic What can do to prevent this You should dissolve your peptide in DMF Many peptides are difficult to work with because of solubility issues Perform the modification reaction with 1 mole equivalent of HyNic and the reaction should be performed in DMF with 2 mole equivalents of TEA triethylamine The peptide should be modified and stay in solution The 4FB oligo should be dissolved in a 50 50 mix of conjugation buffer and DMF Perform the conjugation reaction in this mixture Recently bought your crosslinking products
7. are listed on our website We offer a variety of formats of this reagent both in kits and as standalone products Solulink s biotin product page provides an overview of our product offerings If you perform a significant amount of biotin labeling one of these other options may work well for you Another consideration we can provide you with a much higher level of technical support than can Pierce As the inventor and developer of this reagent technology and having years of experience with custom conjugations and product development we can better assist you with your projects and help you optimize biotin labeling for any of your applications Also we can offer discounts to you for volume purchases either in kit format or in any custom packaging for your company What s the difference between All in One kits and other antibody labeling kits All in One kits are the only kits that allow you to isolate virtually 100 pure antibody conjugate without HPLC FPLC column purification The All in One Kits used for antibody labeling are the only products of their kind that convert 100 of antibody to conjugate making purification easy with the Solulink mini purification spin columns All in One kits are the only kits for making high yielding pure high quality antibody conjugates Do I need to remove Tris from my buffer prior to labeling my antibody The amines in a glycine or Tris based buffer WILL interfere with optimal labeling of the antibody wit
8. binding capacity means greater capture using less bead mass Less bead mass for your immobilization assays results in better assay capture and increased savings solulink Why do you offer 2 sizes of beads Which one do I choose We have 2 sizes of beads to fit the needs of specific applications Some customers need a uniform sized bead The MagnaLink beads offer this For MOST applications however this is not necessary Some microarray applications might require a certain size as well The larger bead will settle faster if that might be an issue in your application Will unlabeled DNA bind nonspecifically to NanoLink streptavidin beads Random DNA sequences may bind to the surface of the magnetic beads but only very weakly if at all The non biotinylated sequences can easily be washed away from the beads The crosslinked streptavidin doesn t prevent binding How do I ensure that all of my beads become labeled with biotinylated biomolecules All of the beads should be attached to antibody if you use a 10 to 20 fold molar excess of biotinylated product Washing away unbound antibody is straightforward you can magnetize the beads and wash them to remove unbound antibody You can repeat this a few times it is not trivial to remove any unbound beads from bound beads Will NanoLink streptavidin beads experience less nonspecific binding NSB of biotinylated nucleic acids or biotinylated proteins than other beads We DO think that
9. do recommend using a blocking agent such as casein We sell a bead block for this purpose NanoLink Streptavidin beads have you tested chemical resistance to urea detergents We haven t performed any specific tests but streptavidin is a pretty stable protein so concentrations of these reagents that are protein friendly would be fine with regards to the streptavidin coated beads Do you have beads for direct binding of antibodies peptides or oligos The NanoLink 4FB beads are used for direct binding of HyNic modified biomolecules To learn more please visit the NanoLink 4FB product page Regarding the MagnaLink Streptavidin beads can the amount of biotin bound to the bead be quantitated using a fluorescent tag Does the bead quench the fluorescence in any way We routinely quantify the biotin binding capacity of our beads using fluorescein biotin However since it is very difficult to ascertain the effect of the bead on quantum yield and other properties of the fluor we avoid this situation by fluorescently quantifying the amount of unbound vs bound bioin fluorophore left in solution By simple subtraction we obtain the amount bound If you use this technique it is always wise to run a control bead without bound streptavidin if possible as well as a no bead control ANTIBODY LABELING KITS Why use ChromaLink Biotin or ChromaLink Digoxigenin DIG labeling kits For maximum sensitivity and reproducibility i
10. min 7 Add an additional 10 50 pL water to the bead solution and continue the incubation for 5 min 8 The washing step is very important Washed the beads 3X each with water ethanol water PBS and Conjugation Buffer 100mM sodium phosphate 150 mM NaCl pH 6 0 in that order using the spin protocol from step 1 9 Check the supernatant to see if there is a significant Az from the Hynic silane add 100 uL of supernatant to 900 uL of Buffer The Azg9 should give a reading no higher than 0 05 10 Bring the beads up in Conjugation Buffer such that the solution is a 20 w v beads in Conjugation Buffer solulink The Hynic modified beads are now ready to be conjugated the 4FB modified biomolecule For large biomolecules proteins and antibodies 20 ug of protein mg of bead is recommended for maximum conjugation Allow the beads to conjugate with the protein overnight at room temperature on a rotor or shaker Can DMF or DMSO be used for dissolving non sulfo crosslinkers Can my resuspended reagent be used just one time or several times after opening Both solvents can be used but either needs to be anhydrous for long term stability of non sulfo linkers in solution Many biology labs have DMSO but not DMF so they can use their own DMSO but cannot store the solution for further use We need to prepare 1 1 antibody protein conjugates using your protein protein crosslinking kit Suggestions Example obtaining antibody 30 kDa protein 1
11. the optimal way to conjugate a peptide and an oligo It would be far more efficient to add the HyNic linker to the peptide during synthesis and the 4FB linker to the oligo during synthesis and we can give quotes for both and simply let them react Our Antibody Oligo kit is optimized in several ways for antibodies Can I use a longer oligo longer than 60 bases in your Antibody Oligo kit In our experience oligos up to 60 bases work best If you use a longer oligo we cannot guarantee success but it should still work for you with some slight modification to the protocol We would suggest that you allow the conjugation reaction to go overnight at 4 C This should improve the efficiency which you might expect to be lower as the length of the oligo increases If want to modify a 3 amino oligo with HyNic would I use the sulfo or regular version You don t need to use the sulfo HyNic Use the standard protocol for S HyNic Use the modified oligo immediately After the coupling reaction antibody oligo we used a Nanodrop ND 1000 to test the absorbance of the coupling products We first tested the absorbance at 354 nm user manual we wanted to confirm if the conjugation was successful We did not observe strong absorbance there so we tested the absorbance of the product The 354 absorbance is small compared to the oligo Az 9 but what you have is reasonable You can quantify the number of oligos attached using the extinction coefficient o
12. thereby alter the stoichiometry of the conjugation reaction For best results always use the highest quality HPLC purified amino oligonucleotide available Note Please be advised that some oligo vendors will not HPLC purify amino modified oligos or in some cases longer oligonucleotide sequences modified or unmodified except as a custom service However some oligo suppliers do offer these services as a standard option Solulink recommends that customers always use HPLC purified amino oligonucleotides in this protocol We recommend requesting a mass spectrum to confirm the final quality when available The mass spectrum confirms percent full length purity as well as molecular weight unambiguous confirmation of amino group As a general rule we do not recommend using crude oligonucleotide preparations to make a conjugate Use barrier pipette tips and good laboratory practices at all times to avoid potential contamination and or cross talk between different oligonucleotide sequences The VivaSpin columns in the kit seem to be clogged It is taking a long time to spin down my oligo The VivaSpin is likely being blocked by solids The oligo solutions should be centrifuged at 20 000 x g for 5 min and then used on the VivaSpin Regarding the antibody oligo conjugation we are interested in conjugating oligos to smaller binding proteins that have a unique cysteine tag Is there a protein oligo kit available for conjugations to cysteines If not do you
13. volume and mass of beads e g 100 ug in 100 pL to fully re disperse the beads into their mono disperse state Never use SDS at any concentration in an attempt to disperse the beads since this abolishes their biotin binding capacity Can NP 40 detergent be used with Solulink s NanoLink streptavidin beads We have never used NP 40 with our streptavidin beads nevertheless this detergent should be completely compatible with our beads The literature is full of references in which NP 40 at a final concentration of 0 1 is used to wash streptavidin beads prior to addition of biotinylated molecules In this regard our streptavidin beads should be no different and fully compatible with the use of this detergent The covalent bond used to immobilize streptavidin on the beads is not affected by the presence of this detergent The only detergent that we definitely know destroys all biotin binding capacity is the denaturing ionic detergent SDS solulink Can I heat the beads to cleave the biotin streptavidin bond As with all streptavidin beads excessive heating will lead to dissociation of the streptavidin tetramer and leaching of monomeric subunits We don t recommend heating the streptavidin beads over 40 C NanoLink Streptavidin beads what pH range are the beads stable The beads are stable over a wide pH range from pH 3 to 9 NanoLink Streptavidin beads have you tested for nonspecific protein adsorption Not specifically but we
14. water or any other buffer will be perfectly suitable for storage for 3 months We want to link a fluorescent dye to a peptide already have the peptide Does Solulink provide this kind of kit solulink For pre made peptides you can use a standard NHS ester fluor to label the peptide We do not offer these as stand alone reagents With plans to conjugate a protein to antibody previous attempts via amine linkage on one end the antibody and maleimide coupling on the other end via a cysteine residue on my protein the results were not optimal More reproducible results and more control of the process is desired specifically with less proteins conjugated to each antibody ideally a 1 1 ratio Considering attachment of the protein cysteine to carbohydrate on the antibody notice you have reagents that can couple to cysteine via maleimide do you have a reagent that oxidizes carbohydrate groups and also a linker that couples to the resultant aldehydes We cannot recommend the scheme proposed The way we typically conjugate through the carbohydrate groups is by oxidation which would give one less control not more and the oxidative product is an aliphatic aldehyde which is not as stable as the aromatic aldehyde We get very controllable reproducible results with our UV traceable chemistry that links through the amino groups Can the Antibody Oligo kit be used to conjugate a peptide and an oligonucleotide The Antibody Oligo kit is not
15. LINKING KITS AND REAGENTS How do I know which linker to put on each of my 2 biomolecules Please check our product selection guide coming soon for a detailed explanation If you would like recommendations we welcome you to contact technical support support solulink com How stable are HyNic and 4FB modified proteins stored in aqueous solution AFB modified proteins are stable at 4 C for up to one year HyNic modified proteins are not stable and should be used the same day they are modified preferably HyNic modified proteins can be stored at 80 C in small single use aliquots in conjugation buffer No need to add glycerol And do not re freeze after used reason for the aliquots Should be good for several months You want the proteins frozen not moving around in solution with possible HyNic amino group interaction How stable are HyNic and 4FB reagents stored in DMF from Solulink These reagents are stable for up to 1 month if stored at 4 C What is the stability of S HyNic if stored at 20 C dessicated Our recommended storage time shelf life is 18 months After that time you can reconstitute one of your vials run on an HPLC and see if you observe a single peak If yes then it s still OK If multiple peaks are observed then some degradation may have occured If my TAT HyNic peptide is dissolved in conjugation buffer should I store at 20 C How long the does the solution stay active What molar excess of TAT H
16. Oligo conjugation kit One question that has come up already for me was what is meant by amino modified for the oligo Would a standard DNA synthesis company know exactly is meant by that or is it something special Starting Amino Oligonucleotide requirements The Antibody Oligonucleotide All in One kit is designed to conjugate any high purity 5 or 3 amino modified oligonucleotide 20 60 nucleotides in length to any solulink monoclonal or polyclonal IgG class antibody The protocol requires a minimum quantity of 10 OD2 9 and a maximum of 40 OD269 units of HPLC purified amino oligonucleotide Solulink recommends that longer oligo sequences e g gt 49 mer be synthesized with a 5 amino group and shorter oligos lt 49 mer with a 3 amino group if the specific application permits Oligonucleotides lt 49 bases can be either reverse phase RP or ion exchange purified IEX while longer oligos gt 49 mer can be IEX or double HPLC purified depending on the specific services offered by the vendor Some vendors offer these purification options on a custom basis while others offer them as a standard service albeit at additional cost Be advised that unpurified 3 amino oligos contain a significant quantity of truncated failure sequences that lead to undesirable conjugation products while unpurified 5 amino oligos contain up to 50 of A20 units in the form of shorter unmodified failure sequences that never form conjugate and
17. ave a peptide reagent that adds our S Hynic linker onto either end of the peptide during synthesis 6 BOC HNA Cat No S 3003 For conjugation of carbohydrates we recommend modifying your amino dots with S Hynic S 1002 and then oxidizing the carbohydrates with 10 mM sodium periodate and reacting the two modified bioconjugates If you have carboxy modified Quantum Dots you will need to activate them with sulfo NHS EDC then quench with 100 mM EDA 100 mM borate pH 8 0 to amino modify them prior to using any of our proprietary linkers What is your silica bead modification protocol 1 Weigh out 100 mg of silica beads 2 Wash the silica beads 3 times with EtOH pellet the bead on a centrifuge for 2 min at 750 x g remove the supernatant Bring the beads up in EtOH and repeat 3 Make a 50 mM solution of Hynic silane 10 mg in EtOH 500 uL add 2 10 uL water to the solution to dissolve any remaining solids This may require intensive vortexing to get the silane into solution 4 Add the Hynic silane solution to the washed bead pellet such that the silane silica ratio is 20 w v 500 uL 5 Vortex the bead sample and incubate at room temperature on a rotator for 30 min Note Check the pH periodically with pH paper and be sure that it doesn t go below pH 7 4 during the incubation steps Bring the pH to above 7 4 with 1 M NaOH if the pH drops 6 Add an additional 2 10 uL water to the bead solution and continue the incubation for 15
18. c group adversely Can the protein oligo kit be used to link a single nucleotide or a 2 or 3 mer oligo to an antibody You would not be able to use our Antibody Oligo kit for this kind of project The shortest oligo we have tested is a 15 mer However we do have a custom conjugation group here that works on special projects We could make a nucleotide with our 4FB linker or a 3 mer with 4FB and conjugate that to a HyNic modified antibody Has your company ever perform peptide hapten conjugations for regulatory studies Is there any method to analyze the peptide hapten stability This is important since if there is not a positive response after the immunization we should know whether the conjugation was stable or not Is there any method to determine the amount of peptide that is conjugated to the hapten Does your company provide a Quality Control QC for the conjugation procedure No we do not have GMP facilities Several clients have had Solulink link small molecules peptides and oligonucleotides to KLH using Solulink s bioconjugation technology and in all cases have isolated antibodies Yes the linkage between the peptide and the KLH is chromophoric and can be quantified spectrophotometrically We can provide whatever data you need including protein concentration and number of peptides KLH bought a protein oligo conjugation kit from your company and found that the recommended length of oligo is up to 100 base bairs However my oli
19. duction is not due to loss of streptavidin but rather blocking of NSB binding sites Can I use BSA as a bead block instead of casein The composition of our bead blocking solution is 5 mg mL Hammarsten grade casein in 25 mM Tris 150 mM NaCl pH 7 4 containing 600 ppm Kathon anti microbial agent and 0 05 anti foam A You CAN use BSA in place of casein but sometimes BSA may contain a small amount of biotin which will bind to the SA If BSA is not a problem with any of your downstream applications then yes it should be fine to use this as a blocking agent Can NanoLink 4FB beads work at high temperatures like 45 C with HyNic modified oligos The 4FB magnetic microspheres are designed to work for Hynic modified small biomolecules such as oligos and peptides You can certainly use the 4FB magnetic microspheres in conjunction with the Hynic modified oligos or Hynic modified peptides The 4FB microspheres work fine at temperatures of 45 C You can even go up to 60 C for up to five minutes and still have good stability The Hynic oligo is not very stable due to polymerization therefore we do not recommend that you use high temperatures on the Hynic modified oligo by itself but once it is reacted with the 4FB the bis aryl hydrazone oligo is very stable in temperatures up to 94 C When looking at NanoLink Streptavidin beads under a microscope I see a significant amount of clumping of aggregates some being 20X larger than a sing
20. f the oligo from the product data sheet molar extinction coefficient at A354 is 29 000 Note the protein does not contribute much to the A260 so it can be ignored unless you want to be very accurate Determine the protein concentration with a BCA assay With those numbers you can accurately determine the number of oligos antibody solulink In the antibody oligo kit wonder if after the purification process part B and C in the user manual all of the unconjugated DNA oligo and antibody should be removed from the system if so can we use the total antibody concentration as the value for the coupling product The protocol is set up so there will be lt 5 free oligo or free antibody We recommend that you analyze your conjugates at least initially by gel electrophoresis We recommend a 5 20 gradient gel developed with Coomassie for protein or silver stain for oligo That will tell you how well the conjugations worked Once I have bound my antigen to my antibody how do I release the antigen from the antibody Release the antigen by elution in SDS reducing sample buffer 10 min 95 C Can I use amino modified RNA or only DNA with your antibody oligo kit You CAN use amino modified RNA oligos they work as well as amino modified DNA oligos For your antibody oligo kit can conjugates be prepared with larger amounts of antibody You can prepare antibody oligo conjugates in larger quantities would recommend buying multiple Ab o
21. fication buffer Small contaminants e g various buffer components under 7 kDa will go into the bead matrix or resin and retained as larger molecules flow through the column This both removes buffer components which might interfere with the reaction and puts the biomolecule of interest into a new buffer Tell me more about the Zeba columns from Pierce website Zeba Spin Desalting Columns contain a proprietary high performance desalting resin that offers exceptional desalting and protein recovery characteristics compared to other commercially available resins Even very dilute 25 pg mL protein samples can be successfully processed to obtain greater than 95 retention removal of salts and other small molecules lt 1 000 MW and good recovery of proteins and other macromolecules gt 7 000 MW Despite claims other commercially available resins perform only solulink satisfactorily in spin columns with sample of high concentration gt 250 ug mL over a narrow window of sample volumes The proprietary Zeba Micro Spin Desalting columns has exceptional protein recovery and desalting characteristics with gt 95 retention of salts and other small molecules of lt 1 000 MW and are recommended for processing compounds gt 7 000 MW What is the purpose of the diafiltration spin columns in many of your kits These columns are used to concentrate antibody solutions These size exclusion columns contain a membrane of MWCO of 5 000 this
22. g small molecules that may be in the serum i e hormones minerals etc This will allow for labeling of large amine containing biomolecules only If you were hoping to biotinylate more than 5 mg of protein then we would recommend bypassing the kit and just buying the ChromaLink reagent and then following the protocol on our website to biotinylate your proteins It will be necessary for you to purchase the Zeba desalting columns from Thermo Scientific We recommend that you make a few modifications to the protocol to account for the fact that you will be working with a variety of different plasma proteins You should determine the protein concentration of your serum solution using a BCA assay Thermo Scientific or the A23 and then use an average protein mw perhaps 70 000 kDa to determine the number of moles of protein in the solution Then add 10 molar equivalents of our ChromaLink Biotin Labeling Reagent All of the proteins should be sufficiently biotinylated with this method We are considering using the above kit to biotinylate a monoclonal antibody provided as ascites fluid which has an IgG concentration of around 7 mg mL was wondering if you could advise whether your product would be suitable for this application and whether any particular purification steps would be required Ascites fluid contains not only your IgG antibody of interest but also a number of other proteins found in the gut of the animal in which it is produced i
23. go is 200 base pairs in length Will the 200 bp oligo work in this kit The protein oligo conjugation kit is to be used with an HPLC purified oligo that has been amine modified If you are doing an amine conjugation to your 200 bp oligo the purification is crucial You will need a significant number of nmol of oligo to perform a successful conjugation You can work with our kit and you might have good success However as this strategy is outside our testing conditions we cannot guarantee that you will have success with this product We recently bought your technetium linker kit The protocols are fairly well explained up until the process of linking technetium in the form m technetium pertechnetate with your HyNic linker We solulink simply want to couple mTc pertechnetate to your HyNic linker already bonded to a backbone substrate Can you direct us to a simple protocol of how to incorporate the mTc pertechnetate into your HyNic protocol http www nucmedbio com article S0969 8051 2803 2900056 8 abstract We want to conjugate 10 mg of each of 2 proteins lyophilized One is 14 kDa and the other is 44 kDa You should obtain the larger Zeba columns from Pierce if you want to perform a larger scale reaction We would suggest performing the conjugation on a much smaller scale Not more than 100 ug of protein In that case the smaller desalting columns would be sufficiently large On the bottom of page 7 of the Protein Protei
24. h HyNic since HyNic reacts with amino groups The All in One kit protocol buffer exchanges your antibody into a phosphate based buffer free of amines so the labeling reaction is optimized The buffer exchange part of the protocol only takes a few minutes and is simple to perform Regarding the Antibody HRP kit Will 0 1 sodium azide and 0 1 gelatin disturb the conjugation The antibody has to be stored at 4 C Are the storage conditions the same after conjugation with HRP You will want to remove any sodium azide which interferes with the modification reaction or gelatin which has Lys The azide can be removed by the desalting column or buffer exchange column included in the HRP kit The gelatin is more of an issue because of its size will not be removed by the buffer exchange size exclusion column Commonly used additives include preservatives such as sodium azide thimerosal protein stabilizers such as BSA or gelatin and or small molecule additives such as glycine or trehalose Protein carriers such as BSA or gelatin must be removed before labeling can proceed Remove and purify away all protein carriers such as BSA or gelatin by affinity or other chromatographic methods re adjust the initial antibody concentration to 1 mg mL or you can ask the antibody vendor to NOT add any stabilizers to your antibody preparation The antibody HRP conjugate should also be stored at 4 C and should be stable for at least one year solulink CROSS
25. he specificity of the beads in their particular application Other researchers require low nonspecific binding with very high binding capacity and don t care about leaching These researchers do want a blocked bead If one is going to capture biotinylated DNA you probably don t need blocked beads pH and detergents turn out to be solulink more important factors for reducing DNA based NSB than any other factors If one is going to capture biotinylated antibody or proteins one should block the beads for optimal results and minimal NSB For some applications pre blocked beads would be a good thing and for others not needed Regarding reduction of binding capacity using blocked beads experiments we have done indicate that binding capacity is reduced for antibodies But the reason for this is because 15 of the binding is NSB to begin with and not related to biotin streptavidin interactions Even after extensive casein blocking our NanoLink Streptavidin beads still bind 250 yg of biotinylated IgG vs 80 ug for Invitrogen s product based on their website per mg So after blocking we still bind 3X more antibody than anybody else Without blocking we capture a great deal more biotinylated IgG but this increased capture is nonspecific binding believe that we have captured up to 400 ug of biotinylated IgG per mg of NanoLink without blocking the beads first So yes you will see a reduction in binding of biotinylated proteins but this re
26. ibody Suggestions One thing to try is to reduce the antibody concentration to around 1 mg mL and use 20 equivalents of ChromaLink Biotin This should help improve or increase the MSR If a precipitate is observed you may be experiencing another situation Some antibodies are very sensitive to lysine modification this could be one of them Charges are being removed when lysine residues are modified thereby affecting solubility of the protein How do dissociate or break the streptavidin biotin interaction Some information below is borrowed from some other vendors as this is a commonly asked question If you do need to remove the captured antigen from the SA beads you might also consider using a cleavable biotinylation reagent Without a cleavable reagent below are some of the common strategies used which are harsh yet necessary to break the very strong bond The streptavidin biotin interaction is the strongest known non covalent biological interaction between a protein and molecule The bond formation between biotin and avidin is very rapid and once formed is unaffected by wide extremes of pH temperature organic solvents and other denaturing agents Unless derivative forms of biotin or modified streptavidin have been adopted for your experiment requiring a specific form and normally gentle way to dissociate biotin from streptavidin often very harsh methods are required to dissociate the biotin from streptavidin which will denature
27. ication Buffer 1 0 M Sodium Phosphate 1 5 M Sodium Chloride pH 7 4 1X concentration 10X PBS 1 0 M Sodium Phosphate 1 5 M Sodium Chloride pH 7 2 1X concentration want to label 10 mg of protein with biotin Suggestions One should purchase reagents separately to save money You can purchase 5 mL Zeba columns from Pierce The 10X Modification Buffer without azide is S 4000 005 This is 5 x 1 5 mL for 50 You can get the ChromaLink reagent in bulk 5 x 1 0 mg if preferred which is B 1007 105 for the water soluble version or 10 mg which is Cat No B 1007 010 The best modification conditions 1 Protein concentration is 5 6 mg mL 2 Normally 6X equivalence of linker used MSR will be 4 4 5 If you want high MSR just increase to 8X or 10X 3 The 5 mL Zeba desalt spin column is good for 500 2 000 pL 4 Use 2 5 mL 1X MB or 1X PBS to pre equilibrate column at 1 000 x g for 2 min three times before desalt desalt at 1 000 x g for 3 min Use 15 mL tube as collection tube Can diluted reconstituted ChromaLink biotin be frozen for storage longer than one day then reused at a later time The diluted biotin reagent should NOT be used after 1 day even if frozen Should be used fresh as hydrolysis can occur The sulfo ChromaLink Biotin reagent is stable for an hour or for the day at most if kept at 4 C but not longer than that Hydrolysis occurs solulink We got a very low MSR when we biotinylated our ant
28. igo synthesis would provide similar information In general for biotinylation of proteins peptides or other amine containing molecules we recommend our product with confidence but for oligos we would just have the oligo synthesized with the biotin group What is the difference between your ChromaLink Kit and your ChromaLink One Shot Kit The ChromaLink One Shot Biotin Kit B 9007 009K 150 biotinylates 100 ug of antibody at 1 mg mL We also have an additional kit the ChromaLink Biotin Labeling Kit B 9007 105K which is more flexible in format and can label up to 5 reactions of between 25 ug to 1 mg per 100 L reaction The reaction solulink conditions were specifically designed to match the concentrations and quantities of most commercially available antibodies We have an application where we want to biotinylate all proteins in a plasma serum sample We will deplete abundant proteins first what Solulink product or kit is best for this application Our ChromaLink Biotin Labeling Reagent will work very well for your protein plasma serum biotinylation The product that we recommend depends on how much you plan to biotinylate Our ChromaLink Biotin Labeling Kit allows for 5 separate reactions of 1 mg of total protein each This kit comes with desalting apparatus with a molecular weight cut off of 7 000 kDa The ChromaLink labeling reagent labels any amino groups so these desalting columns will remove any amine containin
29. inimal amount of DMF 33 to keep the linker soluble but above that amount there is no affect on the reaction It is important to dilute the reaction with buffer or water prior to desalting on the VivaSpin column Can the hydrazone bond formed between HyNic and 4FB be broken Yes with addition of hydroxylamine We have references for this procedure With 100 mM NH2OH and 100 mM aniline cleavage is quantitative within 8 hours want to conjugate protein A 43 kDa to antibody IgG 150 kDa We have both cross linkers C6 SANH HyNic linker and C6 S 4FB linker My protein is very sensitive to pH buffer composition temperature and solvent It s very risky to incubate at room temperature for a long time How many fold excess of linker would be suitable for this protein Is it ok to incubate this reaction at 4 degrees C for 2 hr for modification Please suggest the antibody IgG 150 kDa amount solulink will use C6 S 4FB crosslinker for the modification of antibody For the conjugation reaction which molar ratio would be suitable for best conjugation of both modified proteins I am not sure it depends upon the amount of modified protein or crosslinker attached in modifying protein The molecular weight of my protein A 43 kDa is approx three times lesser than the antibody IgG 150 kDa Is it ok to incubate the conjugation reaction for 12 hr at 4 degrees C with pH near 7 for best conjugation All of our modification data is at
30. ith the Solulink linkers 4FB and HyNic you can easily quantitate the number of linkers you add to each biomolecule As with labeling for maximum sensitivity and reproducibility it is important to avoid too many linkers which leads to reduced immune reactivity or too few linkers which leads to weak signal or unconjugated material Quantitative linkers allow for more control and reproducible steps during the conjugation reaction How does the PEG4 PFB react with amines The product S 1034 010 PEG4 PFB Long Chain Crosslinker does react with primary amine groups and does have a PEG4 linker arm When looking at the structure on the product data sheet you notice the penta solulink fluoro phenyl group This is the leaving group and the mechanism is very similar to the NHS ester mechanism The nitrogen of the amine group attacks the carbonyl carbon adjacent to this potential leaving group and with some rearrangement along with participation of water the phenyl group leaves Does your chemistry work with Quantum Dots Our chemistry works very well with Qdot Nanocrystals If you are using amino dots to link peptides would recommend using sulfo S 4FB S 1008 to modify the Dots and then use Hynic to modify your peptide Our linking chemistry links through the e amino group on the lysine S Hynic S 1002 or through a thiol group on a cysteine MHPH S 1009 allowing for direct modification of your peptide after synthesis We also h
31. le bead Are the beads OK to use We have sometimes noticed aggregation with NanoLink SA beads but have recently Nov 2010 modified our SOP to greatly reduce clumping issues This can occur from the way the beads are prepared and the SA crosslinked If you have a sonicator available you might try performing a 1 minute sonication to disrupt aggregates After a 30 60 sec sonication treatment there was very little clumping observed mostly single beads Since the beads are very concentrated to begin with 10 mg mL the recommendation is that once diluted that you sonicate your sample for 30 60 sec prior to your application Beads obtained after November 2010 should experience very little clumping Magnetic beads blocking buffers elution conditions and resolving clumping issues solulink Blocking Buffers For immobilization of proteins we recommend using a casein buffer We have a bead block buffer available Cat No S 4023 025 Elution conditions We recommend using a glycine buffer pH 2 8 for elution You should collect the antigen in a neutral phosphate buffer to limit exposure to the harsh pH conditions Clumping Solutions To obtain a mono disperse population of NanoLink Streptavidin beads these procedures will help Vortex the beads for 1 minute in their original container to resuspend the pellet 2 Remove a 10 pL aliquot mass equivalent to 100 ug of beads from the dispersed suspension using a P 10 pipette
32. ligo kits we have volume discounts because they are much easier to use than any alternative Alternatively you can use the more general protein oligo kit which can conjugate mg amounts amount of antibody But this kit does not contain the unique purification step that comes in the Ab oligo kit and requires HPLC purification of the final antibody oligo conjugate want to use a cleavable 4FB linker with your antibody oligo kit Which is the cleavable linker If you use the Ab oligo kit this comes with the S 4FB crosslinker Instead of using the crosslinker in the kit you may use an alternative 4FB crosslinker to attach to your oligo the S SS 4FB crosslinker Cat No S 1037 The Excel spreadsheet that comes with the kit the calculator can tell you how much of the reagent to use once resuspended You would then be able to cleave the Ab oligo linkage which is a disulfide bond How does the Solulink Antibody Oligonucleotide kit differ from what Olink offers The following list describes how Solulink s All in One Antibody Oligonucleotide Conjugation Kit differs from what Olink offers Olink s antibody oligo product is a component of their in situ IHC PLA product It includes the specific oligos for that product It conjugates only 10 ug of antibody There is no indication of the purity i e conversion to conjugate There is no purification step It is not available as a standalone product I m interested in using your Antibody
33. n conjugation kit manual you are directed to add a large amount of HyNic in DMF because the amount of protein is large Would suggest making 5 mg mL stock solutions You should scale this reaction down significantly modifying just 100 ug of each protein Would suggest modifying the larger protein with 15 equivalents of HyNic and the smaller protein with 5 equivalents of 4FB 4FB is a much more efficient crosslinker am trying to conjugate an antibody to another protein with S HyNic and S 4FB In Table 1 MSR of S HyNic saturates to 8 or 9 MSR of S 4FB saturates to 26 or 28 What makes the difference between those two values Does S HyNic or S 4FB or conjugate product have any charges that affect the conjugation efficiency Different linkers have varying modification efficiencies 4FB modifies much more efficiently than does HyNic 4FB is very hydrophobic and binds nonspecifically initially to protein hydrophobic regions then is in close proximity to and reacts with Lys residues very efficiently HyNic does have a protonated group pyridine and does not crosslink to the biomolecule with the same efficiency as 4FB The charge on the HyNic perhaps contributes to it s lower modification efficiency but does not affect its conjugation efficiency to 4FB In working with the protein oligo kit am curious about the use and amount of DMF suggested The DMF is required so that the S 4FB is soluble in the reaction mixture The reaction requires a m
34. n this case mouse An antibody in this form is not acceptable for conjugation because the biotinylation reaction targets amino acids on the antibody that would also be found in the other proteins Your biotin streptavidin assay would then be detecting not only the activity of the IgG antibody but also the activity of all the other proteins you have biotinylated For biotinylation reactions we recommend only using purified antibodies You could use our kit to biotinylate a secondary antibody to your Monoclonal Anti Tyrosine Hydroxylase antibody which would detect antigen binding of the monoclonal But if you biotinylate the ascites fluid you will just have a nonspecific binding mess giving falsely high antigen binding results Our biotinylation kit comes with desalting columns but is not equipped for specific purification of antibodies What is the difference between the Solulink biotin labeling kit and the Pierce biotin labeling kit Solulink is the inventor and manufacturer of the NHS chromogenic biotin labeling reagent ChromaLink Biotin Many of the biotin labeling kits from Pierce use an NHS Biotin reagent WITHOUT the chromogenic fluorophore Using a chromogenic biotin reagent offers MANY advantages you can use simple UV quantitation to determine biotin incorporation with no need for the HABA assay an additional assay solulink required to determine the extent of biotin incorporation There are several other advantages as well which
35. ors affect absorbance measurements or the affinity purification steps This shouldn t be a problem MISCELLANEOUS What is the shelf life for your kits How long are they stable If stored at the recommended temperature our kits should be stable for at least 18 months How do I dissociate my antigen from my antibody Because the binding is so tight harsh methods are typically used to dissociate an antibody from its target antigen One can use 0 1 M glycine 0 5 1 Triton X 100 at pH 2 8 or boil in protein lysis buffer at 95 100 C 5 10 min Some vendors market a less harsh method of separating the two biomolecules Pierce sells a gentle Ag Ab elution buffer We sometimes notice a precipitate in your 10X buffers Are the buffers OK to use The 10X conjugation buffer and 10X modification buffer contain a high concentration of salt which can precipitate out of solution and form crystals at room temperature and especially when stored at 4 degrees This is normal and occurs frequently There is nothing wrong with the buffers You can heat the 10X solution to 40 degrees and vortex to get all of the salt crystals back into solution Then you can use the buffer to make your 1X dilution What is the purpose of the desalting columns used in many of your kits Zeba spin columns These columns are size exclusion columns with a MWCO of 7 kDa These are used to exchange buffers to put the biomolecule to label in the correct buffer such as a modi
36. our NanoLink streptavidin coated beads might provide you with lower nonspecific protein binding to your biotinylated biomolecule compared to competing products for 2 reasons 1 Because of the highly crosslinked streptavidin using our unique patented chemistry there is less polymer surface area on the bead exposed for proteins or nucleic acids to adhere to This in itself should reduce NSB for you Our extensively crossliked streptavidin is the reason the biotin binding capacity of these beads is so high 2 And because of the high biotin binding capacity you can use FEWER beads in your assay again reducing nonspecific protein binding Do I need to block my beads How Depending on your application Solulink recommends blocking streptavidin beads If you are capturing non phosphorylated proteins we recommend blocking with Bead Blocking Solution Cat No S 4023 025 If you are capturing DNA or RNA we recommend blocking with yeast tRNA salmon sperm or herring sperm DNA at 10 mg mL if the presence of blocking DNA or RNA are not a factor in your downstream application Some bead manufacturers use beads that are blocked Are blocked beads better to use Many researchers do not want blocked beads because the blocking protein can and does leach some Not much but some It is not covalently linked so some leaching occurs Some researchers do not want any protein leaching since this would interfere for example with protein sequencing and t
37. products and would like to know if you have any conjugation kits that have been tested successfully with protein lipid or protein carbohydrate combinations We have successfully used our technology to link proteins to lipids and carbohydrates The lipid or carbohydrate in question has to be modified with an amine however and the chemistry to do this isn t trivial If you have a lipid or carbohydrate which is already amine modified then one of our kits will make the conjugation quite easy to perform If you do not have amine modified biomolecules this sort of conjugation is often times performed by our custom conjugation group We have done this before and he can give you tips suggestions or a quote for a service to do this for you Regarding conjugation of a HyNic peptide and 4FB oligo we are mostly concerned with getting all of our 4FB sites on our DNA conjugated to protein not with the efficiency of protein conjugating to the oligo The instructions for conjugation seem to suggest a 2 1 ratio of DNA to protein to obtain 95 protein conjugation But if we are more interested in the DNA then I assume our ratio should be less I am wondering how much less Also if we would like to store some of the HyNic peptide for later use is it stable if stored in buffer at 802C If your main goal is to conjugate all of the 4FB oligo then you may want to use a ratio closer to 1 1 The number of oligos a given protein can carry is largely governed by i
38. ps on Asp or Glu residues The kit has been developed so that only 3 8 biotins are labeled onto the antibody There are about 50 Lys residues on any given antibody so if you are only modifying three of them the probability that you are hitting the antigen binding site is low While we cannot guarantee that the antigen binding site is not being labeled rendering it inactive we rarely see a case where labeling causes the antibody to become inactive How do I remove or denature the non biotinylated strand of dsDNA bound to streptavidin beads Remember only double stranded DNA that has biotin on one of the strands can be denatured Typically it will be a PCR product that was amplified with one biotinylated primer and the other must be non biotinylated If both strands are biotinylated you cannot easily remove the DNA from the beads without destroying the streptavidin completely 1 After immobilization of biotinylated PCR DNA place the 1 5 mL tube on a magnetic rack for 2 min to collect the beads onto the side of the tube 2 Using a pipette carefully remove and discard the clarified supernatant taking care not to dislodge the pellet 3 To denature the non biotinylated strand add 200 pl of 100 mM NaOH freshly prepared from 10N stock to the beads and vortex to resuspend the beads Incubate for 60 sec to denature the non biotinylated strand 4 Place the microfuge tube back on the magnetic rack for 2 min 5 Carefully remove and discard
39. r linking chemistry links through the amino group on the lysine or through a thiol group on a cysteine allowing for direct modification of your peptide after synthesis We also have a peptide reagent that adds our S Hynic linker onto either end of the peptide during synthesis Our chemistry then requires a separate modification of the antibody protein with a traceable linker that corresponds to S Hynic This linker is S 4FB The resulting conjugate that forms when the two linkers come together has a hydrazone chromaphore that allows the end user to know exactly how many peptides are on the antibody This chemistry provides several advantages when compared to SMCC namely it is more reproducible it is traceable and it reacts specifically preventing undesirable polymer formation How do I remove excess 4FB after peptide modification Reverse phase HPLC would be the best way to remove excess 4FB post modification Can I use a lower amount of antibody say 50 pg for the Antibody Oligo conjugation kit Yes you can The percent yield will be lower but the antibody will be linked to the oligo We strongly suggest that you use 100 ug of antibody The kit was optimized for this amount Is the 4FB moiety heat stable on oligos There should be no problem with using this for PCR before conjugation to my HyNic peptide Yes it is heat stable We have had users successfully amplify 4FB oligos and link the amplicons am interested in your crosslinking
40. room temperature While modification at 4 degrees may work we cannot guarantee success For the modification we would suggest 15 equiv at 2 mg mL protein A concentration We suggest 10 equiv at 2 mg mL For the conjugations we recommend a 1 1 mol mol ratio However we suggest trying 1 1 5 and 1 2 antibody protein A ratios on an initial small scale 10 ug reactions that can be analyzed by PAGE Use TurboLink buffer in the solution to optimize the conjugation reaction Should add 4FB or HyNic to my amino modified oligo We have found that our Hynic oligos have a tendency to dimerize if not used immediately after modification If you are planning on immobilizing the oligo on 4FB beads if the Hynic oligo is added to the 4FB magnetic microspheres immediately after desalting then you shouldn t see too much dimerization We have used this scheme before with success For these reasons for typical reactions such as antibody oligo conjugations you want to modify your oligo with 4FB not with HyNic What are the compositions of your modification and conjugation buffers Both the modification and conjugation buffers are provided at a 10X concentration Each is provided as 5 x 1 5 mL vials So 7 5 mL x 10 75 mL of 1X buffer Modification Buffer 1 0 M Sodium Phosphate 1 5 M Sodium Chloride pH 7 4 Conjugation Buffer 1 0 M Sodium Phosphate 1 5 M Sodium Chloride 0 05 Azide pH 6 0 What are the advantages of using a quantifiable linker W
41. s lt 50 kDa 3 4 for 60 100 kDa proteins and 4 6 for antibodies This has to do with the percentage of Lys residues present and how many charges are removed which can and will affect the protein coming out of solution solulink would like to know if the biotin labels affect the binding of the antigen to the antibody For example if all the asp and glu carboxyl groups are changed on a protein then will the antibody still recognize it and be able to bind Biotinylation probably won t affect binding to an antibody although there are certainly risks If you are using a monoclonal antibody they might be more site specific especially with the N terminus which will likely be modified by our biotinylation reagent Additionally the larger your antigen the less likely biotinylation will inhibit binding Really the only way to know is to try it we have a lot of experience labeling proteins and antibodies and this really is the best way to modify without changing the biomolecule too much Other reagents will not be as controllable and use more harmful chemistries that could harm your protein would recommend using lower biotinylation levels depending on the size of your antigen would recommend not adding more than 10 molar equivalents and it should work out fine This reagent specifically labels amine groups on Lysine residues The beauty of our linking reagent is that there is no reduction or oxidation step that would alter the hydroxyl grou
42. solulink FAQ Database February 2011 e MAGNETIC BEADS e ANTIBODY LABELING KITS e CROSSLINKING KITS AND REAGENTS e MISCELLANEOUS MAGNETIC BEADS Tell me about the types of beads that you offer We currently offer two different types of magnetic beads NanoLink is a less uniform bead mixture which has 150 and 800 nm diameter beads and MagnaLink is a very uniform bead with a 2 8 um diameter Both bead types are made with magnetite encapsulated by a non styrene polymer surface Unless you are particularly concerned with uniformity e g for NMR flow cytometry etc the NanoLink beads are particularly suited for high capacity capture of oligos peptides and antibodies Both bead types come in three different varieties streptavidin coated 4FB linker coated and amine coated As a custom service we can get other bead types for you and link them to 4FB or streptavidin And we can add a linker with an extended spacer but currently offer only streptavidin coated 4FB coated and amino beads as catalog products NanoLink Bead size bimodal non uniform distribution 150 nm to 800 nm Density 1 5 g cm Magnetite content Fe30 40 w w Magnetic moment 25 emu g electromagnetic units per gram MagnaLink Bead size 2 8 micron uniform distribution Density 1 9 g cm Magnetite content Fe30 50 w w Magnetic moment N A What are the advantages of using high biotin binding capacity beads Higher
43. t is important to avoid over labeling which leads to reduced immunoreactivity or under labeling which leads to a weak signal ChromaLink Biotin and DIG Labeling Kits are the only kits which enable quantification of the incorporated label with a simple UV scan of the labeled protein No more HABA assays By simply measuring the biotin or DIG labeled protein at 280 and 354 nm and inserting the values into an easy to use calculator provided with the kit you can determine the exact number of biotins or DIG on your antibody MSR Consistent quantifiable labeling provides you with consistent reliable and reproducible results in your assays Can all any antibodies and or proteins be labeled with ChromaLink reagents solulink Yes All proteins and antibodies may be labeled with ChromaLink Biotin or DIG labels These products label primary amino groups on lysines found in proteins A cysteine labeling ChromaLink biotin reagent is also available Cat No B 1012 105 How do I remove excess biotin from my reaction If you are using the One Shot kit or the standard ChromaLink kit this is done in step 6 of the procedure One Shot protocol or the second buffer exchange step standard kit using the Zeba spin columns included in the kit 2nd Buffer Exchange remove excess labeling reagent What is the difference between 10X modification buffer and 10X PBS There is only a minor pH difference between these 2 buffers 10X Modif
44. the clarified supernatant 6 Neutralize any residual base by washing the beads 2X with 500 uL of 0 1 M Tris HCL 150 mM NaCl solulink 7 The single stranded DNA immobilized on the beads is now ready for hybridization by addition of denatured complementary target DNA in a suitable hybridization buffer Therefore if you were to use the biotinylated oligo with our streptavidin coated magnetic beads you would easily be able to capture the complementary strand of the biotinylated oligo and then remove it with 100 mM NaOH which would quickly break the hydrogen bonds leaving the DNA backbone intact This type of de hybridization scheme is much easier to buffer exchange than the detergent method Can I biotinylate carbohydrates and lipids with your kit Our reagent will biotinylate any amine containing biomolecule If your carbohydrate or lipid has been modified with amines this kit will work for you There are some disclaimers though The kit Cat No B 9007 105k comes with desalting apparatuses with a molecular weight cut off of 7 000 kDa Therefore if your biomolecules are smaller you may be better off buying our reagent Cat No B 1007 105 by itself and using a different desalting apparatus diafiltration spin column or cassettes with smaller molecular weight cutoffs will work well Also while carbohydrates may be desalted with the Zeba desalting columns we provide the hydrophobic nature of your lipid may lead to losses when desalted
45. the streptavidin A couple of these methods are discussed below For biotinylated proteins boil the beads in 0 1 SDS or SDS PHAGE buffer for 3 min Our antibody is very dilute e g 0 25 mg mL What concentration of antibody should use for your ChromaLink kit How many equivalents of ChromaLink Biotin should use We typically recommend an antibody concentration of at least 1 mg mL for the biotinylation reaction If infeasible to concentrate your IgG four fold then increase the molar equivalents to 30 so that you can have a final MSR between 5 and 8 If you have a method of concentrating your antibody e g Vivaspin Diafiltration filters Cat No S 4004 010 would recommend that first then adding 20 equivalents of the ChromaLink reagent Can I use avian IgY antibodies for ChromaLink Biotin labeling While the presence of lysine groups on an antibody is paramount with regards to chicken IgY it is too heavily_glycosylated to work as well as bovine IgG with our kits Although IgY will be labeled or modified with biotin to some extent the sugars can interfere with binding to streptavidin in downstream applications So the glycosylation can be an issue when working with IgY antibodies in terms of downstream applications where attachment to streptavidin is involved What is the optimal MSR for biotinylation of my protein or antibody The optimal biotin MSR depends on the MW of the protein We suggest 2 3 biotins for protein
46. think it would work to use the MHPH Maleimide HyNic crosslinker and the S 4FB crosslinker for the amine oligo to cys protein conjugation When reading the protocol for the MHPH it does not say that the protein needs to be reduced is this correct We do not have a protein oligo kit made specifically for protein cysteine oligo conjugations but your planned strategy is exactly right You would want to use the S 4FB crosslinker to modify your oligonucleotide which has an amino group at the 3 or 5 end and crosslink that product to an antibody or protein modified with HyNic The way to conjugate your protein at a Cys residue instead of a Lys residue to HyNic is with our MHPH reagent then react the two modified biomolecules together to get your conjugate It is recommended to add TCEP tricarboxyethylphosphine to the MHPH modification reaction which reduces any disulfide bonds but does not react with the maleimido group Recommended conditions are adding 3 5 mol equiv of TCEP mol protein during the modification step We also recommend conjugating the HyNic modified protein immediately solulink Can you explain the mechanism of the magnetic affinity purification in your Antibody oligo kit This information is proprietary Is there any problem with using the Antibody oligo kit to conjugate an amino oligo to which fluorescein or Cy5 is already attached to the antibody guess this would either be from the perspective of whether the flu
47. ts surface area which is roughly proportionate to its molecular weight In our experience an antibody has no difficulty conjugating to multiple oligos in the 20 30 nucleotide range Smaller proteins would do better with a 1 1 or perhaps 2 1 solulink DNA protein ratio Particularly if you don t mind having free protein in the crude reaction then this is not a problem would add our TurboLink catalyst buffer to drive the reaction to completion Regarding the AFB on the oligonucleotides we order the amino derivitized oligo and then incorporate the 4FB post synthetically using our 4FB HNS linker This process is quantitative in our experience The ultimate purity of the oligo in terms of full length 4FB modified is therefore dependent on the incorporation efficiency of the amino linker Therefore it is important to remember that although you might be adding 2 molar equivalents of oligo to your protein for conjugation you re really adding only 1 1 equivalent of 4FB oligo The unmodified oligo cannot conjugate and will remain in the conjugation reaction Regarding the peptide it is most stable as a lyophilizate If you have an analytical balance you can weigh out 1 2 mg to dissolve for your studies and store the rest lyophilized at 80 C Barring that possibility you may dissolve the whole tube and aliquot it out to usable portions and store at 80 C Avoid freeze thaw of the peptide as this will cause it to precipitate and may affect the HyNi
48. varies by the type of kit Molecules less than this size will pass through the membrane and larger molecules will be retained These columns can be used to concentrate antibodies prior to labeling or modification What is the purpose of the Q spin filters used in your HRP kit These are ion exchange columns with a positive charge on the resin surface The pH of one of the solutions in the kit is used to alter the charge on the conjugated antibody above the pl of the antibody the antibody HRP complex will have more negative charges and will bind to the resin At this pH the HRP is still positively charged a higher pl and will pass through the column thereby separating the free HRP from the Ab HRP conjugate Then the pH is changed again below the pl of the antibody thereby increasing the positive charge on the Ab HRP conjugate such that it passes through the column The same strategy is used in the PE kit My DMF appears to be cloudy or have a precipitate in it Is it OK to use DMF is a clear colorless liquid The gray grainy particles sometimes brownish orange depending on the batch and supplier are the molecular sieves that help keep it anhydrous
49. with these columns The chromaphore will still absorb strongly at 354 nm but our kit helps you determine the MSR molar substation ratio based on the concentration of the protein usually determined by A280 You could assume 100 recovery of your biomolecule during the biotinylation reaction in order to determine the amount of biotinylation but it won t be as accurate as with protein using the Azs absorption If you have a better way of determining the concentration of your carbohydrate or lipid then you would be able to more accurately determine the biomolecule biotin ratio l am currently using your ChromaLink Biotin Kit want to purify the labeled antibody that have made by Protein G purification methods which will require the use of an elution buffer namely 0 1 M Glycine at pH 2 or 3 which is quickly neutralized with 1 M Tris pH 8 or 9 Do you know what the effect of the glycine is on the biotin linkage Does glycine affect the chromophore The bond that is formed between the antibody and the ChromaLink reagent is an amide bond which is very stable in low pH The ChromaLink chromaphore will not be affected by the low pH glycine buffer Your protein G purification will not have any negative effect on the biotinylation of your protein l lost approximately 65 of my biotinylated protein after the second Zeba spin column What happened It is unlikely that the protein is still on the column as the resin excludes molecules with
50. yNic should I use for antibody conjugation After conjugation can purification be done by using a desalting column or by dialysis What kind of buffer is best for antibody TAT storage Once in solution the peptide should either be used ASAP or frozen at 80 C Be sure to aliquot the peptide solution into usable portions prior to freezing to avoid the need to freeze thaw TAT HyNic peptide conjugation A 3 5 mole excess of TAT HyNic peptide should be used Purification can be performed with a diafiltration coilumn with a 5 kDa MWCO or by dialysis Storage buffer should be PBS with 0 5 azide How does one determine how many 4FB molecules are bound to my biomolecule 2 Hydrazinopyridine is an aromatic hydrazine reagent used to quantify the molar substitution ratio MSR of 4FB modified proteins or other biomolecules This aromatic hydrazine reacts with 4FB modified biomolecules and forms a quantifiable hydrazone absorbance signature at 350 nm How does one determine how many HyNic molecules are bound to my biomolecule 2 Sulfobenzaldehyde is a water soluble aromatic aldehyde reagent used to quantify the molar substitution ratio MSR of HyNic modified proteins or other biomolecules This aromatic aldehyde reacts with HyNic modified biomolecules and forms a quantifiable hydrazone absorbance signature at 390 nm solulink Does Solulink use SMCC Pierce to conjugate a peptides to proteins We do not use a single bifunctional linker like SMCC Ou
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