Pierce NHS-Activated Magnetic Beads - Fisher Scientific
Contents
1. aggregation and loss of binding activity can result from using these methods Pierce Biotechnology PO Box 117 815 968 0747 www thermoscientific com pierce 3747 N Meridian Road Rockford IL 61105 USA 815 968 7316 fax Thermo SC hEING Pare e Estimate the amount of protein coupled to the magnetic beads with a protein assay e g Thermo Scientific Pierce 660nm Protein Assay Product No 22660 and 22662 and subtract the amount of flow through protein from the loaded protein To measure the amount of protein on the bead directly use the Thermo Scientific Pierce Micro BCA Protein Assay Product No 23235 see Tech Tip 75 from our website e For coupling antibodies to magnetic beads ensure the antibody storage solution does not contain a protein stabilizer e g BSA gelatin which inhibits coupling of the antibody to the beads Protein stabilizers can be removed using the Thermo Scientific Pierce Antibody Clean up Kit Product No 44600 see Tech Tip 55 from our website For best results buffer exchange the antibody into 50mM borate pH 8 5 e g Thermo Scientific BupH Borate Buffer Packs Product No 28384 e Primary amine containing buffers e g Tris and glycine inhibit coupling of protein to the magnetic beads Remove primary amine containing buffer using dialysis e g Thermo Scientific Slide A Lyzer G2 Dialysis Cassettes 10K MWCO 3mL Product No 87730 or desalting e g Thermo Scientific Zeba Spin Desalting Columns 7K2. the protocol to the KingFisher Flex or KingFisher 96 Instrument from an external computer See the BindIt Software User Manual for detailed instructions on importing protocols 3 Set up plates according to Table 4 Table 4 Pipetting instructions for the IP protocol using Microtiter Deep Well 96 Plates Plate Plate Name Content Volume Time Speed Beads 25uL 1 Bind 2 hour Slow Antigen Sample for IP 500uL 2 Wash 1 Binding Wash Buffer 500uL 30 seconds Slow 3 Wash 2 Binding Wash Buffer 500uL 30 seconds Slow 4 Wash 3 Ultrapure Water 500uL 30 seconds Slow 5 Elution 1 Elution Buffer 100uL 5 minutes Slow 6 Elution 2 Elution Buffer 100uL 5 minutes Slow 7 Tip Plate air corer ati mi 10 seconds Fast 4 Select the protocol using the arrow keys on the instrument keypad and press Start See the KingFisher Flex or KingFisher 96 Instrument User Manual for detailed information 5 Slide open the door of the instrument s protective cover 6 Load plates into the instrument according to the protocol requests placing each plate in the same orientation Confirm each action by pressing Start 7 After sample processing remove the plates as instructed by the instrument s display Press Start after each plate Press Stop after removing all of the plates Notes e The low pH elutions must be neutralized by adding 10uL of Neutralization Buffer for each 100uL of eluate directly to each well immediately after incu
3. HS Activated Magnetic Beads e Visit www thermoscientific com kingfisher for information on KingFisher Products e Inthe U S A purchase KingFisher Supplies from VWR Outside the U S A contact your local Thermo Fisher Scientific office to purchase KingFisher Supplies Related Thermo Scientific Products 88828 Pierce Direct Magnetic IP Co IP Kit 88802 Pierce Protein A G Magnetic Beads 1mL 88803 Pierce Protein A G Magnetic Beads 5mL 88804 Pierce Protein A G Magnetic IP Co IP Kit 88805 Pierce Crosslink Magnetic IP Co IP Kit 88816 Pierce Streptavidin Magnetic Beads 1mL 88817 Pierce Streptavidin Magnetic Beads 5mL 88821 Pierce Glutathione Magnetic Beads 4mL 88822 Pierce Glutathione Magnetic Beads 20mL 23235 Micro BCA Protein Assay Kit 21030 Gentle Ag Ab Binding and Elution Buffer Kit 44600 Pierce Antibody Clean up Kit Tween is a trademark of Croda International Plc This product Product is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale as set forth in the Product documentation specifications and or accompanying package inserts Documentation and to be free from defects in material and workmanship Unless otherwise expressly authorized in writing Products are supplied for research use only No claim of suitability for use in applications regulated by FDA is made The warranty provided herein is valid only when used by properly trained individu
4. INSTRUCTIONS Thermo Pierce NHS Activated Magnetic Beads 88826 88827 2320 0 Number Description 88826 Pierce NHS Activated Magnetic Beads 1mL supplied at 10mg mL in N N dimethylacetamide DMAC 88827 Pierce NHS Activated Magnetic Beads 5mL supplied at 10mg mL in N N dimethylacetamide DMAC Note Before using refer to the product label for the expiration date Storage Upon receipt store at 4 C Product shipped with an ice pack Table of Contents Tmt GU CHIOM AEAEE AEEA cased A AA vakcck T T TE cots digsannecosloustebesatatasedivncads Wesuleeesebieetasettatoas 1 Important Product Informations ennen ia EEEa dvet cavatedea cues bacssnnt ectaaesd cbevucesatnesvinda ss E EAEE AEE A i 1 Procedure for Protein Immobilization ssis noinein aeni a a a i a aE a E AEE SONER EAE E A aE iei 2 Procedure for Immunoprecipitation c css scsccececsenescevocvsoevsssescsesceesstescesasersecaveneesosecbaenersnsvsteosssessesseeeetecberorseseessvessesrsessessnsees 5 A Manual Antigen Immunoprecipitation ee eee eeccesecesecesecseecaeecaeeeeeeeeeeeeeeeceaeeaecaecaecsaecaaecaeesaeseaeeeeeeeeeeeeeeseaeesaees 5 B Automated Antigen Immunoprecipitation 0 0 eee cee cseeeseeeeeeeeeeeeeesecsecaececeseceaecsaecsaecaaecaeesaeseeeeeaeeesseeeeeseaeeaees 5 FPOUDIESHOOUI GS s cscatsisaissc cbsieeds ie nE ees E EREE E EEE E EEEREN EE TEE EEEE ENE ETE 6 Additional Informationen ne aE EE EEE SEEE SEENE E E EEE EE EES EN EE EEE EEE TEE EEEE 7 Related Thermo Sc
5. MWCO 0 5mL Product No 89882 e The coupling efficiency of protein to the magnetic beads varies depending on the specific protein Typically 0 1 2 0mg mL of protein produces optimal protein coupling however optimize the concentration for each specific protein As a reference binding capacities of different proteins are listed in Table 2 Table 2 Binding capacity of proteins with different molecular weights on the Thermo Scientific Pierce NHS Activated Magnetic Beads Molecular weight Binding capacity of NHS beads Protein kDa ug mg of bead IgG 150 50 Streptavidin 53 n Protein A G 50 2 Protein L 36 2 Note Results will vary depending on the number of accessible primary and secondary amines Procedure for Protein Immobilization Note The following coupling procedure is for 300uL of magnetic beads in a 1 5mL microcentrifuge tube scale the procedure as needed A Additional Materials Required e Wash Buffer A ice cold 1mM hydrochloric acid e Coupling Buffer 5 0mM borate pH 8 5 Product No 28384 or other amine free buffer pH 7 9 e Protein Solution 0 1 2 0mg mL in Coupling Buffer For proteins already in solution completely remove primary amine containing buffer e g Tris or glycine using desalting or dialysis e Quenching Buffer 3M ethanolamine pH 9 0 e Storage Buffer Coupling Buffer with 0 05 sodium azide e 1 5mL microcentrifuge tubes e Ultrapure water e Wash Buffer B 0 1M glycine pH 2 0 For M
6. als Unless otherwise stated in the Documentation this warranty is limited to one year from date of shipment when the Product is subjected to normal proper and intended usage This warranty does not extend to anyone other than the original purchaser of the Product Buyer No other warranties express or implied are granted including without limitation implied warranties of merchantability fitness for any particular purpose or non infringement Buyer s exclusive remedy for non conforming Products during the warranty period is limited to replacement of or refund for the non conforming Product s There is no obligation to replace Products as the result of i accident disaster or event of force majeure ii misuse fault or negligence of or by Buyer iii use of the Products in a manner for which they were not designed or iv improper storage and handling of the Products Current product instructions are available at www thermoscientific con pierce For a faxed copy call 800 874 3723 or contact your local distributor 2011 Thermo Fisher Scientific Inc All rights reserved Unless otherwise indicated all trademarks are property of Thermo Fisher Scientific Inc and its subsidiaries Printed in the USA Pierce Biotechnology PO Box 117 815 968 0747 www thermoscientific com pierce 3747 N Meridian Road Rockford IL 61105 USA 815 968 7316 fax 7
7. anual Coupling e Magnetic stand e g Thermo Scientific Magnabind Magnet for 6 x 1 5mL microcentrifuge tubes Product No 21359 Pierce Biotechnology PO Box 117 815 968 0747 www thermoscientific com pierce 3747 N Meridian Road Rockford IL 61105 USA 815 968 7316 fax 2 Thermo SC MENGE Pare For Automated Coupling eee es oh 10 11 12 13 14 15 16 17 18 19 C KingFisher Flex System with 96 deep well head Product No 5400630 or KingFisher 96 System Product No 5400500 Thermo Scientific Microtiter Deep Well 96 Plate V bottom polypropylene 100 1000yL Product No 95040450 KingFisher Flex 96 Tip Comb for Deep Well Magnets Product No 97002534 Manual Procedure for Coupling and Blocking Equilibrate protein solution and magnetic beads to room temperature Note To ensure homogeneity mix the magnetic beads thoroughly before use by repeated inversion gentle vortexing or using a rotating platform Place 300uL of magnetic beads into a 1 5mL microcentrifuge tube Place the tube into a magnetic stand collect the beads and discard the supernatant Add 1mL of ice cold Wash Buffer A into the tube and gently vortex for 15 seconds to mix Place the tube into a magnetic stand collect the beads and discard the supernatant Note Immediately proceed with adding the protein solution Add 300uL of protein solution into the tube and vortex for 30 seconds Incubate the tube for 1 2 hours at room temperat
8. bation e If fewer than 96 wells are used fill the same wells in each plate For example if using wells Al through A12 use these same wells in all plates e Combine the Tip Comb with a Deep Well 96 Plate See the instrument user manual for detailed instructions Troubleshooting Problem Possible Cause Solution Low coupling efficiency Primary amine containing buffer was not Dialyze or desalt sample to completely remove completely removed before coupling Tris and glycine Protein addition was delayed Immediately mix protein with beads after washing Protein is not soluble in Molecule was hydrophobic Dissolve protein in coupling buffer containing coupling buffer up to 20 DMSO Beads aggregate during Proteins on bead surface adhered to tube After blocking add 0 05 detergent e g the coupling process plastic Tween 20 Detergent to the water wash and the Storage Buffer Part C Note Do not use detergent in the coupling step Pierce Biotechnology PO Box 117 815 968 0747 www thermoscientific com pierce 3747 N Meridian Road Rockford IL 61105 USA 815 968 7316 fax 6 Additional Information Visit www thermoscientific com pierce for additional information relating to this product including the following e Frequently Asked Questions e Tech Tip 43 Protein stability and storage e Tech Tip 55 Remove BSA and gelatin from antibody solutions using Melon Gel e Tech Tip 75 Measure protein bound to Pierce N
9. ientific Products vicssecesssisescisseecdesstasnonesenteeand buovesedetevdecossnvenstenducestabsesntedaeceseonederensndvcssteabeocsvaceesecvedenededsedueceoss 7 Introduction The Thermo Scientific Pierce NHS Activated Magnetic Beads covalently immobilize proteins for the affinity purification of antibodies antigens and other biomolecules The activated magnetic beads contain N hydroxysuccinimide NHS functional groups which react with primary amines on proteins or other molecules to form stable amide linkages The coupling reaction is performed in an amine free buffer at pH 7 9 The beads are manually removed from the solution using a magnetic stand or by automation using an instrument such as the Thermo Scientific KingFisher Flex System Automated instruments are especially useful for large screening of multiple samples Table 1 Characteristics of Thermo Scientific Pierce NHS Activated Magnetic Beads Composition N hydroxysuccinimide NHS functional groups on a blocked magnetic bead surface Magnetization Superparamagnetic no magnetic memory Mean Diameter um nominal Density 2 0g cm Bead Concentration 10mg mL in DMAC Binding Capacity gt 26ug of rabbit IgG mg of beads Important Product Information e Magnetic beads are moisture sensitive To protect the beads cap the bottle immediately after removing the slurry and wrap lab film around the cap before storing at 4 C e Do not centrifuge dry or freeze the magnetic beads Bead
10. nstrument keypad and press Start See the KingFisher Flex or KingFisher 96 Instrument User Manual for detailed information 5 Slide open the door of the instrument s protective cover 6 Load the Tip Plate plate 11 and press Start The instrument places the Tip Comb onto the magnet head 7 Remove the Tip Plate 8 Load plates 1 8 into the instrument according to the protocol requests place each plate in the same orientation Confirm each action by pressing Start 9 After sample processing through plate 8 the instrument will pause and instruct to remove each individual processed plate while simultaneously loading the remaining three plates plates 9 11 For example remove plate 1 and load plate 9 into that position Confirm each action by pressing Start 10 After sample processing remove the plates as instructed by the instrument display Press Start after each plate Press Stop after removing all of the plates Notes Load ice cold 1mM Wash Buffer A in plate 2 immediately before instrument loading to ensure the buffer remains cold If using fewer than 96 wells fill the same wells in each plate For example if using wells Al through A12 use these same wells in all plates Combine the Tip Comb with a Deep Well 96 Plate See the instrument user manual for detailed instructions Pierce Biotechnology PO Box 117 815 968 0747 www thermoscientific com pierce 3747 N Meridian Road Rockford IL 61105 USA 815 968 7316 fax 4 Pr
11. ocedure for Immunoprecipitation A Additional Materials Required 1 5mL microcentrifuge tubes Binding Wash Buffer Tris buffered saline TBS Product No 28360 containing 0 05 Tween 20 Detergent and 0 5M NaCl Low pH Elution Buffer 0 1M glycine pH 2 2 5 Antibody for immunoprecipitation Antigen sample for immunoprecipitation e g cell lysate Neutralization Buffer High ionic strength alkaline buffer such as a 1M phosphate or 1M Tris pH 7 5 9 Optional Protease inhibitor cocktail e g Thermo Scientific Halt Protease Inhibitor Single Use Cocktail EDTA free Product No 78425 For Manual IP Magnetic stand e g MagnaBind Magnet for 6 x 1 5mL Microcentrifuge Tubes Product No 21359 For Automated IP C KingFisher Flex System with 96 deep well head Product No 5400630 or KingFisher 96 System Product No 5400500 Thermo Scientific Microtiter Deep Well 96 Plate V bottom polypropylene 100 1000uL Product No 95040450 KingFisher Flex 96 Tip Comb for Deep Well Magnets Product No 97002534 Manual Antigen Immunoprecipitation Add 25uL 0 25mg of antibody coupled magnetic beads to a 1 5mL centrifuge tube Place tube in a magnetic stand collect the beads and discard the supernatant Dilute antigen sample for immunoprecipitation to 1 2mg mL using Binding Wash Buffer Add 500uL of diluted antigen sample to the tube containing antibody coupled magnetic beads and incubate for 1 2 hours at room temperature on a ro
12. r KingFisher 96 Instrument The protocol can be modified according to your needs using the Thermo Scientific BindIt Software provided with the instrument 1 2 Enter the Protein Coupling protocol from Table 3 into the BindIt Software on an external computer Transfer the protocol to the KignFisher Flex or KingFisher 96 Instrument from an external computer See the BindIt Software User Manual for detailed instructions on importing protocols Pierce Biotechnology PO Box 117 815 968 0747 www thermoscientific com pierce 3747 N Meridian Road Rockford IL 61105 USA 815 968 7316 fax 3 Thermo SC MEIN Pre 3 Set up plates according to Table 3 Table 3 Pipetting instructions for the Coupling and Blocking protocol using the Microtiter Deep Well 96 Plates Plate Plate Name Content Volume Time Speed 1 Beads NHS Activated Beads 300uL Collect Beads 2 Wash 1 Wash Buffer A ImL 10 seconds Slow 3 Coupling Protein Sample in Coupling 300uL 1 2 hours Slow Buffer 4 Wash 2 Wash Buffer B ImL 10 seconds Slow 5 Wash 3 Wash Buffer B ImL 10 seconds Slow 6 Wash 4 Purified Water ImL 10 seconds Slow 7 Quench Quenching Buffer ImL 2 hours Slow 8 Wash 5 Purified Water ImL 30 seconds Slow 9 Wash 6 Storage Buffer ImL 30 seconds Slow 10 Storage Storage Buffer 300uL Release Beads 11 Tip Plate KingFisher Flex 96 Tip Comb for Deep Well Magnets 4 Select the protocol using the arrow keys on the i
13. tator or mixer Gently vortex the beads every 10 15 minutes during incubation to ensure the beads remain in suspension Collect the beads with a magnetic stand remove the unbound sample and save for analysis Add 500uL of Binding Wash Buffer to the tube and gently mix Collect the beads on a magnetic stand and discard the supernatant Repeat this step one time Add 500uL of ultrapure water to the tube and gently mix Collect the beads on a magnetic stand and discard the supernatant Add 100uL of Elution Buffer to the tube Incubate for 5 minutes at room temperature on a rotator or mixer Magnetically separate the beads and save the supernatant containing the target antigen Note One elution may be sufficient however optimization is required for each system Repeat Step 8 and combine the two eluates To neutralize the low pH of the solution add 10uL of Neutralization Buffer for each 100uL of eluate Automated Antigen Immunoprecipitation Note The following protocol is designed for use with the KingFisher Flex or KingFisher 96 Instruments The protocol can be modified according to your needs using the BindIt Software provided with the instrument 1 Enter the Direct IP protocol from Table 3 into the BindIt Software on an external computer Pierce Biotechnology PO Box 117 815 968 0747 www thermoscientific com pierce 3747 N Meridian Road Rockford IL 61105 USA 815 968 7316 fax 5 Thermo SC MENG Pare 2 Transfer
14. ure on a rotator During the first 30 minutes of the incubation vortex the tube for 15 seconds every 5 minutes For the remaining time vortex the tube for 15 seconds every 15 minutes until incubation is complete Note If required incubate overnight at 4 C Collect the beads with a magnetic stand and save the flow through Add ImL of Wash Buffer B to the beads and vortex the tube for 15 seconds Place the tube into a magnetic stand collect the beads and discard the supernatant Repeat Steps 9 and 10 one time Add ImL of ultrapure water to the beads and vortex for 15 seconds Place the tube into a magnetic stand collect the beads and discard the supernatant Add 1mL of Quenching Buffer to the beads and vortex the tube for 30 seconds Incubate the tube for 2 hours at room temperature on a rotator Place the tube into a magnetic stand collect the beads and discard the supernatant Add ImL of purified water to the tube mix well collect the beads with a magnetic stand and discard the supernatant Add ImL of Storage Buffer to the tube mix well collect the beads with a magnetic stand and discard the supernatant Repeat this wash two additional times Add 300uL of Storage Buffer to the beads mix well and store at 4 C until ready for use Note The final concentration of the protein coupled magnetic beads is 10mg mL Automated Procedure for Coupling and Blocking Note The following protocol is designed for use with the KingFisher Flex o
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