NanoLink™ 4-FB Magnetic Beads User Manual
Contents
1. 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 Wavelenath nm Figure 5 Absorbance scans of peptide solutions 500uM incubated with NanoLink 4FB Magnetic Microspheres Negative controls duplicates included incubation with similar amino modified magnetic beads or no beads Note the reduction in the optical density at 280 nm vs negative control reactions Similarly another negative control using the same peptide sequence without HyNic did not bind to the beads data not shown IV Immobilization of Antibodies to NanoLink 4FB Magnetic Microspheres A Introduction Immobilization of an antibody to NanoLink 4FB magnetic microspheres is similar to the process of immobilizing a peptide This process is illustrated in Figure 6 NanoLink 4FB Magnetic Microspheres HyNic Antib ody 4FB oe O R lt N G N n W ale H o Polystyrene Z N N syr iad r y N Outer core hydrophilic polymer surface Incubate iron magnetite central layer surface Figure 6 Immobilization of HyNic modified antibody to NanoLink 4FB Magnetic Microspheres However in practice antibody immobilizations are more complex because PepLink peptides are small synthetic molecules that come pre activated with HyNic and are ready for immobilization to NanoLink 4FB magnetic microspheres 10 In contrast antibodies are large complex proteins that come in a variety of different initial formulations They also require m2. gt ENT Al TR Version 08 30 2012 NanoLink 4 FB Magnetic Beads User Manual Catalog M 1001 Technical Manual l Product Description II Applications Ill Immobilization of PepLink Peptides to NanoLink 4FB Magnetic Microspheres Introduction Washing NanoLink 4FB Magnetic Microspheres Materials Required Peptide Immobilization Protocol Summary Peptide Immobilization Data MOO IV Immobilization of Antibodies to NanoLink 4FB Magnetic Microspheres A Introduction Important Considerations Prior to Antibody Immobilization Materials Required Initial Antibody Concentration Concentrating Antibodies Buffer Exchange of Concentrated Antibody Modification of Antibody using S HyNic Buffer Exchange of HyNic Antibody Antibody Immobilization Washing NanoLink Antibody Magnetic Microspheres Summary Antibody Immobilization Data ACUT nNMoOD I Product Description NanoLink 4FB magnetic microspheres are uniform polymer encapsulated nanometer sized super paramagnetic particles activated with 4 formyl benzamide functionalities 4FB see Figure 1 The aromatic aldehydes are introduced to the hydrophilic amino modified surface by reaction with SoluLink s sulfo succinymidly 4 formylbenzoate Sulfo S 4FB The high surface area and low non specific binding of NanoLink 4FB magnetic microspheres are ideal for covalent immobilization of aromatic hydrazine modified antibodies peptides DNA RNA oligonucleotides
3. 1M NaH2PQ 1 5M NaCl pH 6 8 SoluLink 4003 005 10x Conjugation Buffer 1M NaH2PO 1 5M NaCl pH 5 0 SoluLink 4002 015 1x Modification Buffer 0 1M NaHPO 0 15M NaCl pH 7 3 1x Conjugation buffer 0 1M NaH2PO 0 15M NaCl pH 6 0 1x PBS 0 1M Phosphate 150mM NaCl pH 7 2 D Initial Antibody Concentration Immobilization requires a minimum mass of 0 25 mg of antibody at a concentration of 2 5 mg ml for each 0 5 mg NanoLink solid phase Antibody preparations come either as solids or liquids The instructions that follow indicate how your particular sample should be processed prior to immobilization Solid Antibody solid form If the initial carrier free antibody to be immobilized is lyophilized solid form then simply re suspend the antibody in sufficient 1x Modification Buffer pH 8 0 to obtain a 2 5 mg ml solution This protocol requires a minimum of 0 25 mg antibody per 0 5 mg of NanoLink A volume of 100 ul 2 5 mg ml is required 0 25 mg for this immobilization protocol If the initial antibody is at 2 5 mg ml in a volume of 100 ul then proceed directly to the Buffer Exchange of Concentrated Antibody section IV F Antibody Solution initial concentration greater than 2 5 mg ml If the initial carrier free antibody is already in solution at a concentration of 2 5 mg ml or greater transfer a volume equivalent to 2 5 mg to a 1 5 ml tube and dilute with 1x Modification Buffer pH 8 0 to obtain 100ul 2 5 mg ml This
4. a BCA protein assay 10 Now that the antibody is at the appropriate concentration proceed to exchange the concentrated antibody solution into modification buffer pH 8 0 Section IV F F Buffer Exchange of Concentrated Antibody Concentrated antibody 2 5 mg ml must be desalted and buffer exchanged prior to modification with S HyNic SoluLink highly recommends the use of Zeba Desalt Spin Columns Figure 8 to desalt proteins This product is available from Pierce Chemical Cat 89882 and is required by our immobilization protocol This rapid spin column is recommended because it does not significantly dilute antibodies during buffer exchange whereas other methods significantly dilute the sample being desalted Figure 8 Zeba desalt spin column 0 5 ml 15 Buffer Exchange Using a 0 5 ml Zeba Spin Column 1 Prepare a 0 5 ml Zeba desalt spin column by removing the bottom closure and loosening the top red cap do not remove cap 2 Place the column in 1 5 ml microcentrifuge collection tubes 3 Centrifuge at 1 500 x g for 1 minute to remove storage solution 4 Place a mark on the side of the column where the compacted resin is slanted upward Place columns in the microfuge with the mark facing outward in all subsequent centrifugation steps 5 Add 300 ul of 1x modification buffer pH 8 0 to the top of the resin bed 6 Centrifuge at 1 500 x g for 1 minute to remove buffer 7 Repeat step 5 and 6 tw
5. protocol requires a minimum of 0 25 mg antibody per 0 5 mg of NanoLink 12 If the initial antibody is at 2 5 mg ml in a volume of 100 ul then proceed directly to the Buffer Exchange of Concentrated Antibody section IV F Antibody Solution initial concentration less than 2 5 mg ml If the initial carrier free antibody is in solution at less than 2 5 mg ml then the antibody solution needs to be concentrated to 2 5 mg ml using a VIVASPIN 500 diafiltration spin filter section IV E Note In our experience under certain conditions antibodies can irreversibly aggregate on the surface of a VIVASPIN 500 filter membrane while they are being concentrated Great care must be taken during concentration to avoid this irreversible aggregation of the antibody The antibody is slowly concentrated w frequent and intermittent mixing of the antibody solution between centrifugation steps Over concentration of the antibody on the membrane surface is to be avoided E Concentrating Antibodies Determine the initial protein concentration of the antibody if unknown using BCA protein assay or other suitable assay e g Bradford assay A minimum of 0 25 mg of total protein is required before concentration can begin To determine if sufficient total mass of antibody is available i e 0 25 mg multiply the initial volume of antibody available for immobilization ml by the initial antibody concentration mg ml see Eq 1 Equation 1 mg ml x m
6. to a final volume of 100 ul Note When transferring or mixing solutions in the concentrator body make sure never to contact or puncture the membrane in the process Never bring a pipette tip in contact with the membrane 3 After loading orient the VIVASPIN 500 spin filter in the centrifuge so that the volume markers on the concentrator body face toward the center of the centrifuge rotor each time 4 Centrifuge for exactly 3 minutes 7 500 x g 5 Check the volume Using a pipette gently and slowly pipette the antibody solution up and down 10 15 times to mix the antibody solution thoroughly within the concentrator body Avoid foaming Mixing avoids aggregation of 14 the antibody on the surface of the VIVASPIN membrane After mixing replace the cap on the concentrator 6 Re orient the spin filter back in the centrifuge and centrifuge for another 3 minutes 7 500 x g 7 Repeat steps 5 and 6 as many times as required until the final volume in the device is 100 ul or less and the sample is concentrated to 2 5 mg ml Note If the final target concentration 2 5 mg ml has been exceeded then add a sufficient volume of 1x modification buffer to the sample to bring the final volume to 100 ul and 2 5 mg ml 8 Carefully transfer the concentrated antibody sample from the concentrator body into a new 1 5 ml microfuge tube 9 Remove a 2 ul aliquot of the concentrated sample 5ug and confirm the protein concentration using
7. Antibody Immobilization In order to successfully immobilize antibody to NanoLink 4FB magnetic microspheres a minimum of 0 25 mg of carrier free antibody no BSA or other carrier is required per half milligram of microspheres We do not recommend immobilizing smaller quantities of antibody to NanoLink If carrier protein is present in your particular antibody preparation the carrier must first be removed using Protein G or Protein A affinity column purification methods Several rapid commercial products are available for this purpose e g NAb Protein G Spin Column from Pierce Chemical Cat 89953 Protein concentrations can be determined using any suitable protein determination assay such as a BCA or Bradford protein assay 11 C Materials Required Zeba Desalting Spin Columns 0 5 ml Pierce Chemical Cat 89882 VIVASPIN 500 Diafiltration Spin Filter Sartorius Cat VS0112 Antibody Required 0 25 mg minimum Molecular grade water 1L Ambion Cat 9932 10 Tween 20 S HyNic SANH 5 mg solid SoluLink Cat S 1002 105 Anhydrous DMF SoluLink Cat S 4001 015 NanoLink 4FB Magnetic Microspheres 1 mg SoluLink Cat M 1001 010 Magnetic tube rack Dynal MPC S or similar BCA Protein Reagents A and B Pierce Chemical Cat 23223 and 23224 Albumin Standard 2mg ml Pierce Chemical Cat 23209 Rainin Pipetman pipettes or similar P 1000 P 200 P 10 Other Required Buffers 10x Modification Buffer
8. B Magnetic Microspheres N Q Oo oa Oo A oO no Oo Antibody binding ug 0 5mg ye oO oO e HyNic IgG Input Antibody 0 5 mg Error bars std deviation triplicates Figure 9 A typical NanoLink 4FB antibody immobilization reaction will immobilize between 12 20 of input mass HyNic lgG per 0 5 mg beads In contrast passive adsorption levels using unmodified IgG are typically 3 5 of input IgG Approximately 65 ug HyNic antibody can be immobilized per half milligram of beads 0 5 mg HyNic lgG input The products offered here are for research use only Any commercial application will require a license from Solulink The Solulink Conjugation System is patented and has multiple patents pending Please contact Solulink for information regarding licensing information Solulink products and methods may be covered by one or more of the following United States patents Nos 6 686 461 6 800 728 7 102 024 7 173 125 7 462 689 and other pending patent applications Information in this manual is subject to change without notice and does not constitute a commitment on the part of Solulink Inc It is supplied on an as is basis without any warranty of any kind either explicit or implied Information may be changed or updated in this manual at any time This document may not be copied transferred reproduced disclosed or duplicated in whole or in part w
9. and or oligosaccharides The paramagnetic properties of these microspheres allow them to be used for the rapid separation of bound from unbound biomolecules using a suitably strong magnet NanoLink 4FB Magnetic Microspheres 4FB lron magnetite central layer Outer core hydrophilic polymer surface Figure 1 NanoLink 4FB Magnetic Microspheres Features High surface area 788 5 nanometer diameter Consistent aromatic aldehyde loading capacities 30 45 nmol mg Encapsulated no exposed iron monodisperse microspheres Fast magnetic response time 25 electromagnetic units 4FB is stable in aqueous solution for months at 4 C Immobilizes approximately 120 ug IgG per mg of solid phase Paramagnetic no residual magnetisim Oooooodo Solids NanoLinkK 4FB magnetic microspheres are packaged at nominally 1 solids 10mg ml as measured using spectroscopic analysis which is set by their optical density at 600nm against a microsphere size standard Aromatic Aldehyde 4FB Functional Capacity The aromatic aldehyde 4FB surface capacity of each lot of particles is quantified by measuring the amount of unbound HyNic peptide OD2 90 left in solution versus an unmodified control microsphere after a 2 hr incubation Refer to Certificate of Analysis for the specific 4FB functional capacity of each lot nmol mg ll Applications NanoLink 4FB magnetic microspheres are a new class of paramagnetic solid phase used to rea
10. dily immobilize aromatic hydrazine modified HyNic biomolecules These reactive microspheres are modified with extremely stable aromatic aldehydes called 4FB that specifically react only with aromatic hydrazine moieties HyNic attached to the surface of any biomolecule NanoLink 4FB magnetic microspheres can be used to immobilize HyNic activated peptides HyNic modified proteins e g antibodies and enzymes The main advantage of SoluLink s HyNic 4FB chemistry is its specificity and stability The aromatic aldehyde 4FB surface reacts only with HyNic modified biomolecules and is insensitive to reaction with other biological functionalities such as NHs SH COOH or OH species The 4FB microspheres exhibit long term storage stability in aqueous solution and remain reactive to hydrazine modified molecules for 6 months at 4 C During reaction of HyNic modified biomolecules to 4FB functional groups the 4FB is immune to hydrolysis or other unwanted side reactions NanoLink 4FB magnetic microspheres are ideal for automated high throughput immobilization processes that use 96 well magnets to affect multiplex binding and separation of defined biomolecules Ill Immobilization of PepLink Peptides to NanoLink 4FB Magnetic Microspheres A Introduction Immobilization of a PepLink peptide to a solid support is as simple as mixing SoluLink s 4FB magnetic microspheres with a hydrazine modified peptide PepLink in an appro
11. e suspension under vacuum using a Speed vac and store at 80 C as a solid 2 Transfer 100 ul 10 mg ml pre washed NanoLink 4FB magnetic microspheres from step B5 into a clean 1 5 ml microfuge tube 3 Add 20 ul of PepLink peptide from step 1 above to the microspheres 4 Place the reaction tube on a platform shaker and allow the immobilization reaction to proceed for 1 hour at room temperature 5 Remove the reaction tube from the shaker and place on a suitable magnetic stand for 2 minutes 6 Remove the supernatant carefully without disturbing the pellet 7 Wash the microspheres using 500 ul 1x Conjugation Buffer 0 05 Tween 20 8 Place the tube on the magnetic stand for 2 minutes and carefully remove the supernatant without disturbing the pellet 9 Repeat step 7 and 8 one additional time Discard the supernatant between washes 10 Re suspend the final microsphere pellet in 100ul buffer of your choice 10mg ml Immobilized peptide on the solid phase is now ready for downstream applications Note Store the microspheres refrigerated but never freeze E Summary of Peptide Immobilization Data Immobilization experiments were carried out using NanoLink 4FB Magnetic Microspheres and PepLink linkable peptides These were used to determine the immobilization binding kinetics of the activated peptide with the solid phase Duplicate reactions were assembled in 1 5 ml reaction tubes containing 500
12. ing to HyNic modified antibody Add 100ul HyNic modified antibody 2 5 mg ml in 1x conjugation buffer to the beads Incubate the microspheres and antibody on a platform shaker for 2 4 h at room temperature 2 Note It is important that agitation be employed to insure efficient immobilization of the antibody and to prevent the microspheres from settling during the coupling procedure 8 After immobilization proceed to washing the microspheres J Washing NanoLink Antibody Magnetic Microspheres 1 Remove the immobilization reaction from the platform shaker 2 Place the tube on a suitable magnetic rack for 2 minutes 3 Carefully and slowly pipette off the supernatant leaving the beads undisturbed 4 Add 1 ml of 1x PBS 0 1 Tween 20 and vortex to wash the microspheres 5 Repeat steps 2 4 two additional times to thoroughly remove any unbound antibody 6 Place the tube on a suitable magnetic rack for 2 minutes 7 Carefully and slowly pipette off the supernatant leaving the beads undisturbed 8 Re suspend the microspheres in 0 5 ml of 1x PBS 0 1 Tween 20 The antibody is now immobilized and the beads are at a final concentration of 1 mg ml 9 Immobilized antibody is now ready for downstream applications 18 K Summary Antibody Immobilization Data Immobilization Characteristics of NanoLink 4FB Microspheres are illustrated in Figure 9 Immobilization of HyNic lgG vs IgG NanoLink 4F
13. ithout the prior written consent of Solulink Inc This documentation is proprietary information and protected by the copyright laws of the United States and international treaties The manufacturer of this documentation is Solulink Inc Gentaur Molecular Products Voortstraat 49 1910 Kampenhout Belgium 19
14. l gt 0 25 mg If the total mass of antibody available is equal to or greater than 0 25 mg but the concentration is less than 2 5 mg ml then proceed to concentrate the antibody Antibody Concentration Protocol Having determined that the total mass of antibody available for immobilization is equal to or greater than 0 25 mg but at a concentration that is less than 2 5 mg ml the sample must be concentrated using a VIVASPIN 500 filter unit as illustrated in Figure 7 Note Dilute antibodies need to be concentrated before HyNic modification because antibodies do not label as efficiently below 2 5 mg ml 13 T lt Concentrator body _ Filtrate tube Figure 7 Spin filter used to concentrated dilute antibody samples Note VIVASPIN 500 filter devices are made to contain and process volumes of 500 ul or less If volumes greater 500 ul need to be concentrated and processed then multiple loading may be required 1 Open the lid of a VIVASPIN 500 filter device 2 Transfer the dilute antibody solution equivalent to 0 25 mg total mass into a VIVASPIN 500 filter device Multiple loadings may be required Examples 1 If the antibody is at 1 mg ml in a volume of 0 25 ml then transfer 0 25 ml antibody solution into a VIVASPIN 500 filter device and concentrate to a final volume of 100 ul 2 If the antibody solution is at 0 50 mg ml in a volume of 1 ml transfer 0 5 ml into a VIVASPIN 500 filter device and concentrate
15. mn where the compacted resin is slanted upward Place column in the microfuge with the mark facing outward in all subsequent centrifugation steps Add 300 ul of 1x conjugation buffer pH 6 0 to the top of the resin bed Centrifuge at 1 500 x g for 1 minute to remove buffer Repeat step 5 and 6 two additional times discarding flow through buffer from the collection tube each time The column is now equilibrated with 1x conjugation buffer and ready for antibody loading Place the equilibrated Zeba column into a new 1 5 ml collection tube and remove cap Slowly apply the entire S HyNic modification reaction to the column Avoid contact with the sides of the column when loading 10 Centrifuge at 1 500 x g for 2 minutes to collect desalted and buffer exchanged HyNic modified antibody Retain the eluate 100 ul 11 Remove a 2 ul aliquot and determine the protein concentration using a BCA protein assay 12 The antibody is now ready for immobilization on washed NanoLink 4FB magnetic microspheres Antibody Immobilization 1 Transfer 50 ul 10mg ml of particles to a clean 1 5 ml microfuge tube 2 Place the 1 5 ml tube on a suitable magnetic tube rack for 2 minutes 17 3 Carefully and slowly pipette off the supernatant leaving the beads undisturbed 4 Remove the tube from the magnetic rack and add 900 ul of 1x Conjugation Buffer 0 05 Tween 20 to the beads Microspheres are now ready for coupl
16. now ready for coupling to HyNic modified peptide i e PepLink peptide Figure 3 Standard commercially available magnetic racks suitable for washing and handling paramagnetic microspheres On the left is a 2 tube magnetic rack and on the right is a 6 tube magnetic rack MPC S both manufactured by Dynal Inc C Materials Required PepLink peptide 0 5 mg solid HyNic activated peptide NH2 terminus NanoLink 4FB Magnetic Microspheres SoluLink Cat M 1001 010 Molecular grade water 1L Ambion Cat 9932 or similar 10 Tween 20 Platform shaker Titer Shaker Lab Line Instruments Vortex mixer Table top microcentrifuge Rainin Pipetman pipettes or similar P 1000 P 200 P 10 Speed VAC optional for long term storage of PepLink Magnetic tube rack Dynal MPC S or similar Invitrogen Corporation 10x Conjugation Buffer 1M NaH2PQ 1 5M NaCl pH 5 0 1x Conjugation Buffer 0 05 Tween 20 0 1M NaH2PO 0 15MNaCl 0 05 Tween 20 pH 6 0 D Peptide Immobilization Protocol Microsphere batch size 1 mg 1 Add 50 ul molecular grade water to 0 5 mg PepLink peptide final HyNic peptide concentration 10 mg ml and vortex until the peptide is completely re suspended Note Once the peptide is dissolved 10 mg ml it is stable for 1 week at 4 C For longer term storage and use 3 months store the 10 mg ml HyNic peptide suspension at 20 C or colder For long term storage gt 1yr dry th
17. o additional times discarding flow through buffer from collection tube 8 The column is now equilibrated with 1x modification bffer and ready for antibody loading 9 Place the spin column in a new 1 5 ml collection tube remove cap and slowly apply 100 ul of concentrated antibody 2 5 mg ml 0 25 mg to the center of compact resin bed Avoid contact with the sides of the column when loading samples 10 Centrifuge at 1 500 x g for 2 minutes to desalt and buffer exchange the sample 11 Retain the desalted and buffer exchanged antibody solution in the collection tube Proceed to modify the antibody with S HyNic IV G G Modification of Antibody using S HyNic SANH 1 Dissolve 5 mg S HyNic S HyNic in 1000 ul anhydrous DMF 2 Add 2 ul of S HyNic DMF solution 20 equivalents to 100 ul of desalted antibody solution 2 5 mg ml and mix well 3 Incubate the reaction at room temperature for 90 minutes 4 Buffer exchange the HyNic labeled antibody reaction into 1x conjugation buffer pH 6 0 using a new Zeba Desalt Spin Column 16 ks ee Er D a Sa 8 9 Buffer Exchange of HyNic Antibody Reaction Prepare a Zeba Spin Column by removing the bottom closure and loosening the top red cap do not remove cap Place the column in 1 5 ml microcentrifuge collection tube Centrifuge at 1 500 x g for 1 minute to remove storage solution Place a mark on the side of the colu
18. odification with S HyNic by the end user before immobilization Initial antibody formulations vary significantly and may include a wide range of protein concentrations different buffers and the inclusion of interfering protein additives Carrier proteins or other additives are often used to stabilize and increase antibody shelf life but these unwanted additives severely interfere with immobilization procedures For these reasons immobilization of an antibody to NanoLink 4FB is considerably more complex than immobilization of a peptide The following steps summarize the antibody immobilization process 1 Concentrate the initial antibody preparation to 2 5 mg ml 15 60 min 2 Exchange the concentrated antibody solution 2 5 mg ml into modification buffer pH 8 0 using Zeba desalt spin column 20 min 3 Modify the antibody solution with S HyNic 90 minutes 4 Exchange the HyNic modified antibody into conjugation buffer pH 6 0 using a Zeba Spin Column 20 min Pre wash NanoLink 4FB magnetic microspheres 5 min Incubate HyNic modified antibody with microspheres 2 4 h Wash NanoLink antibody magnetic microspheres to remove unbound antibody 15 min N OO A great deal of time can be saved if the antibody being immobilized is already at the required concentration of 2 5 mg ml Concentration of the antibody is often the most time consuming hands on step in the overall process B Important Considerations Prior to
19. priate conjugation buffer This process is diagrammed in Figure 2 NanoLink 4FB Magnetic Microspheres PepLink Linkable Peptides O R lron magnetite central layer a N N NN a O H a NION N H ee H A Outer core PepLink peptide J hydrophilic polymer surface Incubate iron magnetite central layer o i Bis aryl hydrazone covalent bond H Cc N H Outer core ags N hydrophilic polymer Pi b LO Si SS H surface PepLink peptide j Figure 2 Diagram illustrating peptide immobilization process on NanoLink 4FB magnetic microspheres B Washing NanoLink 4FB Magnetic Microspheres Note standard peptide immobilization protocol requires 1 mg of microspheres 1 Re suspend NanoLink 4FB magnetic microsphere as supplied in their original container by vortexing or pipetting them vigorously to fully disperse the particles If necessary residual aggregation can be eliminated by brief sonication 10 min 2 Transfer 1 mg of microspheres to a 1 5 ml microfuge tube For example 100 ul is equal to 1 mg of particles 3 Place the tube on a suitable magnetic rack for 2 minutes Figure 3 4 Carefully and slowly pipette off the supernatant leaving the beads undisturbed 5 Remove the tube from the magnetic rack and re suspend the microspheres in 100 ul of 1x Conjugation Buffer containing 0 05 Tween 20 Vortex to mix well 6 One milligram of NanoLink 4FB microspheres is
20. uM PepLink linkable peptide S Tag M W 2030 and 0 5 mg pre washed NanoLink 4FB Magnetic Microspheres re suspended in 100 uL 1x conjugation buffer 0 05 Tween 20 pH 6 0 Control reactions duplicates consisted of 500 uM PepLink peptide in 100 uL 1x conjugation buffer 0 05 Tween 20 pH 6 0 without beads or with 0 5 mg amino modified beads Reactions were carried out on a platform shaker at room temperature At the indicated time points Figure 4 solutions were magnetically separated and bead supernatants assayed by ODz g0 A drop in the OD 2go as the peptide bound to the solid phase was converted into nmol of PepLink bound to the solid phase vs time Figure 4 25 J P Negative control nmol peptide bound per 0 5 mg NanoLink 0 10 20 30 40 50 60 Time minutes Figure 4 Binding kinetics of a PepLink peptide S tag to NanoLinK 4FB Magnetic Microspheres A drop in ODz o vs time measures immobilization kinetics Negative control bead supernatants retain their original OD2g9 In a similar experiment Figure 5 spectral scans 220 420 nm of the bead supernatants were taken after 1 h Negative controls included amino modified microspheres and no microspheres Extended overnight reactions indicate that reactions are complete after 1 h Data not shown oD No microsphere control Neg control gt NanoLink NH Neg control a o t owe sola AFB Mivhetic
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