Pierce Direct Magnetic IP/Co
Contents
1. The final concentration of antibody will be 5ug 100uL and the final concentration of Borate Buffer will be 0 067M Note Adjust the antibody solution for antibody amounts other than Sug per IP 4 Add 100uL of prepared antibody solution to the beads Gently mix and incubate on a rotating platform for 30 60 minutes at room temperature vortex the beads every 10 15 minutes during incubation to ensure that the beads remain in suspension 5 Collect the beads with a magnetic stand Remove the flow through and save for analysis 6 Add 100uL of Elution Buffer and gently vortex or invert to mix Collect the beads on a magnetic stand Remove and discard the supernatant Repeat this wash once Note The Elution Buffer removes non covalently bound antibody 7 Add 500uL of Quenching Buffer to the beads Gently mix and incubate on a rotating platform for 30 60 minutes 8 Collect the beads with a magnetic stand Remove and discard the supernatant 9 Prepare 0 5mL of Modified Borate Buffer for each IP reaction by diluting 25uL of IP Lysis Wash Buffer with 475uL of Borate Buffer prepared from a Thermo Scientific BupH Borate Buffer Pack contents dissolved in a final volume of 500mL ultrapure water 10 Add 500uL of Modified Borate Buffer prepared in Step 9 to the tube and gently vortex or invert to mix Collect the beads on a magnetic stand Remove and discard the supernatant 11 Add 500uL of IP Lysis Wash Buffer and gently vortex or invert to mix2. Pierce Protein A G Magnetic Beads 5mL Pierce Protein A G Magnetic IP Co IP Kit Pierce Crosslink Magnetic IP Co IP Kit Pierce Streptavidin Magnetic Beads 1mL Pierce Streptavidin Magnetic Beads 5mL Pierce Glutathione Magnetic Beads 4mL PO Box 117 7 Rockford IL 61105 USA 815 968 0747 815 968 7316 fax www thermoscientific com pierce 88822 Pierce Glutathione Magnetic Beads 20mL 23235 Micro BCA Protein Assay 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 individuals 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 im
3. minutes Transfer supernatant to a new tube for protein concentration determination and further analysis Manual Procedure for the Pierce Direct Magnetic IP Co IP Kit A Additional Materials Required Magnetic stand e g Thermo Scientific Magnabind Magnet for 6 x 1 5mL microcentrifuge tubes Product No 21359 1 5mL microcentrifuge tubes Ice cold 1mM hydrochloric acid Coupling antibody Antigen sample for IP Ultrapure water 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 MENG Pare B Coupling Antibodies to the NHS Activated Magnetic Beads Note The following protocol is optimized for coupling 5ug of antibody to 25uL of beads but can be used for coupling 2 10ug of antibody This procedure can be scaled up to prepare larger quantities of antibody coupled beads 1 Vortex the bottle of NHS activated magnetic beads to obtain a homogeneous suspension Add 25uL of beads into a microcentrifuge tube Place the tube on a magnetic stand and collect the beads for 1 minute Remove and discard the storage solution 2 Add 400uL of ice cold 1mM HCI to the tube and gently vortex to mix for 5 seconds Collect the beads on a magnetic stand Remove and discard the supernatant 3 Prepare antibody solution by diluting antibody stock into 10uL of Borate Buffer 0 67M and adding ultrapure water to bring the final volume to 100uL
4. 7730 or desalting e g Thermo Scientific Zeba Spin Desalting Columns 7K MWCO 0 5mL Product No 89882 For optimal results use an affinity purified antibody Although serum may be used the antibody specific for the antigen of interest may comprise only 1 2 of the total IgG in the serum sample resulting in low antigen recovery The IP Lysis Wash Buffer has been tested on representative cell types including but not limited to the following cell lines HeLa Jurkat A431 A459 MOPC NIH 373 and U20S Typically 10 HeLa cells yield 10mg of cell pellet and 3ug uL or 300ug when lysed with 100uL of IP Lysis Wash Buffer To minimize protein degradation include protease inhibitors e g Thermo Scientific Halt Protease Inhibitor Single Use Cocktail EDTA free Product No 78425 in the preparation of cell lysates The IP Lysis Wash Buffer is compatible with Thermo Scientific Pierce BCA Protein Assay Product No 23225 Optimal time for low pH elution is 5 minutes if the desired amount of antigen is not obtained then a second 5 minute low pH elution may be performed to remove additional bound antigen 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 Procedure for Mammalian Cell Lysis A Additional Materials Required Phosphate buffered saline PBS 100mM sodium phosphate 100mM NaCl pH 7 2 Product No 28372 Cultured cells
5. Collect the beads on a magnetic stand Remove and discard the supernatant C Antigen Immunoprecipitation 1 Dilute the lysate solution see the Procedure for Mammalian Cell Lysis Section with IP Lysis Wash Buffer to 1 2mg mL 2 Add 500uL of diluted lysate solution to the tube containing the antibody coupled magnetic beads and incubate for 2 hours at room temperature on a rotator or mixer Gently vortex the beads every 15 30 minutes during incubation to ensure the beads stay in suspension 3 Collect the beads on a magnetic stand Remove the unbound sample and save for analysis 4 Add 500uL of IP Lysis Wash Buffer to the tube and gently vortex or invert to mix Collect the beads on a magnetic stand Remove and discard the supernatant Repeat this wash once 5 Add 500uL of ultrapure water to the tube and gently vortex or invert to mix Collect the beads on a magnetic stand Remove and discard the supernatant 6 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 To neutralize the low pH add 10uL of Neutralization Buffer for each 100uL of eluate Note If the desired amount of antigen is not obtained then a second 5 minute low pH elution can be performed to recover additional bound antigen Pierce Biotechnology PO Box 117 815 968 0747 www thermoscientific com pierce 3747 N Meridian Road Rockfor
6. Elute bound antigen Important Product Information 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 Do not centrifuge dry or freeze the magnetic beads Bead aggregation and loss of binding activity may result from using these methods 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 on our website see Tech Tip 75 Measure protein bound to Pierce NHS Activated Magnetic Beads 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 Remove protein stabilizers with the Thermo Scientific Pierce Antibody Clean up Kit Product No 44600 on our website see Tech Tip 55 Remove BSA and gelatin from antibody solutions using Melon Gel 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 8
7. INSTRUCTIONS Thermo Pierce Direct Magnetic IP Co IP Kit 88828 2321 1 Number Description 88828 Pierce Direct Magnetic IP Co IP Kit contains sufficient reagents to perform 40 reactions using 25uL of magnetic beads Kit Contents Pierce NHS Activated Magnetic Beads 1mL supplied at 10mg mL in N N dimethylacetamide DMAC IP Lysis Wash Buffer 2 x 50mL pH 7 4 0 025M Tris 0 15M NaCl 0 001M EDTA 1 NP40 5 glycerol Elution Buffer 5mL pH 2 0 Neutralization Buffer 0 5mL pH 8 5 Quenching Buffer 25mL 3M ethanolamine pH 9 0 Borate Buffer 1mL 0 67M BupH Borate Buffer Pack 0 05M sodium borate pH 8 5 when dissolved in a final volume of 500mL of ultrapure water Lane Marker Sample Buffer Non reducing 5X 5mL 0 3M Tris HCl 5 SDS 50 glycerol lane marker tracking dye pH 6 8 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 IMO MUCH ON xo 2cse cc cade tees cetxcoesesevec ssces vacestessasanced E E soe eeued soe butadeess ceeseeoseeaes eaeeracenederecieneseevs aesiteeas 1 Procedure SUMMARY eroso aon neei reae caacecee cosndstdeancdseveuelesactues E E AE EAE E a E EEA GEO see EEEE ia 2 Important Product Informa NO Masesa eodeni aeaa ae an aa a a aa oa EEE aa aa Ee ces EEEa AEEA EEA AE E A aeS aeia 2 Procedure for Mammalian Celll LySiS eicecien irase ipsae enisi seadsetesstestssestvecsdatesecssdgecesnseta
8. Protocol I Lysis of Cell Monolayer Adherent Cultures Carefully remove culture medium from confluent cells Wash the cells once with PBS Add ice cold IP Lysis Wash Buffer to the cells see Table 2 and incubate on ice for 5 minutes with periodic mixing Table 2 Suggested volume of IP Lysis Wash Buffer to use for different standard culture plates Plate Size Surface Area Volume of IP Lysis Wash Buffer 100 x 100mm 500 1000uL 100 x 60mm 250 500uL 6 well plate 200 400uL per well 24 well plate 100 200uL per well Transfer the lysate to a microcentrifuge tube and centrifuge at 13 000 x g for 10 minutes to pellet the cell debris Transfer supernatant to a new tube for protein concentration determination and further analysis Protocol II Lysis of Cell Suspension Cultures Centrifuge the cell suspension at 1000 x g for 5 minutes to pellet the cells Discard the supernatant Wash cells once by suspending the cell pellet in PBS Centrifuge at 1000 x g for 5 minutes to pellet cells Add ice cold IP Lysis Wash Buffer to the cell pellet Use 500uL of IP Lysis Wash Buffer per 50mg of wet cell pellet i e 10 1 v w If using a large amount of cells first add 10 of the final volume of IP Lysis Wash Buffer to the pellet and pipette the mixture up and down to mix Add the remaining volume IP Lysis Wash Buffer to the cell suspension Incubate lysate on ice for 5 minutes with periodic mixing Remove cell debris by centrifugation at 13 000 x g for 10
9. d IL 61105 USA 815 968 7316 fax 4 Automated Procedure for the Pierce Direct Magnetic IP Co IP Kit A B Additional Materials Required KingFisher Flex System with 96 deep well head Product No 5400630 or KingFisher 96 System Product No 5400500 Thermo Scientific Microtiter Deep Well 96 Plates V bottom polypropylene 100 1000uL Product No 95040450 KingFisher Flex 96 Tip Comb for Deep Well Magnets Product No 97002534 1 5mL microcentrifuge tubes Ice cold 1mM HCl Coupling antibody Antigen sample for IP Ultrapure water Automated Protocol for Antibody Coupling and Antigen Immunoprecipitation Note The following protocol is designed for use with the KingFisher Flex or KingFisher 96 Instrument The protocol can be modified according to your needs using the Thermo Scientific BindIt Software provided with the instrument Note Lysate solution in the protocol is from the Procedure for Mammalian Cell Lysis Section ile 2 Enter the Direct Immunoprecipitation protocol from Table 3 into the BindIt Software on an external computer Transfer 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 Set up plates according to Table 3 Table 3 Pipetting instructions for the automated protocol using Microtiter Deep W
10. ell 96 Plates Plate Plate Name Content Volume Time Speed NHS Activated Beads 25uL 1 Beads Collect Beads Ice cold 1mM HCl 175uL 2 HCI Wash Ice cold 1mM HCl 400uL 5 seconds Slow 3 Coupling Antibody Sample 100uL 30 minutes Slow 4 Elution Buffer Wash 1 Elution Buffer 100uL 10 seconds Slow 5 Elution Buffer Wash 2 Elution Buffer 100uL 10 seconds Slow 6 Quench Quenching Buffer 500uL 30 minutes Slow 7 Wash 1 Modified Borate Buffer 500uL 30 seconds Slow 8 Wash 2 IP Lysis Wash Buffer 500uL 30 seconds Slow 9 Bind Lysate solution 500uL 2 hours Slow 10 IP Wash 1 IP Lysis Wash Buffer 500uL 30 seconds Slow 11 IP Wash 2 IP Lysis Wash Buffer 500uL 30 seconds Slow 12 IP Wash 3 Purified Water 500uL 30 seconds Slow 13 Elution Elution Buffer 100uL 5 minutes Slow 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 PENG Pare 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 the Tip Plate plate 14 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
11. igen binding Perform IP and washes using an alternative buffer e g 0 5 CHAPS in TBS Low amount of recovered protein Protein degraded Add protease inhibitors Not enough magnetic beads were used for capture Increase the amount of magnetic beads used for capture Sample had an insufficient amount of target protein Increase amount of antigen sample Recovered protein was inactive Elution conditions were too stringent Use a milder elution buffer e g Thermo Scientific Gentle Elution Buffer Product No 21034 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 NHS 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 88826 88827 88802 88803 88804 88805 88816 88817 88821 Pierce Biotechnology 3747 N Meridian Road Pierce NHS Activated Magnetic Beads 1mL Pierce NHS Activated Magnetic Beads 5mL Pierce Protein A G Magnetic Beads 1mL
12. it includes optimized buffers that minimize nonspecific binding and maximize antigen yield Lane marker sample buffer is included for preparing samples for SDS PAGE analysis The beads are manually removed from the solution with a magnetic stand or by automation using an instrument such as the Thermo Scientific KingFisher Flex System Automated instruments are especially useful for large scale screenings of multiple samples 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 MENGE Pare 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 1um nominal Density 2 0g cm Bead Concentration 10mg mL in DMAC Binding Capacity gt 26g of rabbit IgG mg of beads Procedure Summary QO Te BR ee ct fi Wash the beads with ice cold 1mM HCI Bind antibody to the beads for 30 60 minutes Wash the beads twice with Elution Buffer Quench the reaction for 30 60 minutes with Quenching Buffer Wash the beads once with Modified Borate Buffer and once with IP Lysis Wash Buffer Incubate cell lysate with antibody bound beads for 2 hours at room temperature or overnight at 4 C Wash the beads twice with IP Lysis Wash Buffer and once with ultrapure water
13. plied 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 com 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 8
14. sample processing through plate 8 the instrument will pause and instruct to remove each individual processed plate while simultaneously loading each of the remaining six plates plates 9 14 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 e Load ice cold 1mM HCI and beads in plates 1 and 2 immediately before loading onto the instrument to ensure minimal hydrolysis of the beads e Neutralize the low pH elutions by adding 10uL of Neutralization Buffer for each 100uL of eluate directly to each well immediately after incubation e 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 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 Dialyze or desalt sample to completely remove Tris not completely removed before and glycine coupling Protein addition was delayed Immediately mix protein with beads after washing Wrong pH for the coupling buffer Use non amine containing buffer at pH 7 9 for the coupling buffer Insoluble protein in Protein was h
15. sensterscenscedassensasecessesttests 3 Manual Procedure for the Pierce Direct Magnetic IP Co IP Kit essseeseeseesseesseesresrsrreresreeresrerreestentrsrenteseeisseeresrnreeresreerese 3 Automated Procedure for the Pierce Direct Magnetic IP Co IP Kit cece csecseeeneeeeeeeeeeeeeeeceseeaecsaecaecseesaeeeneseaeeeeeeeeeas 5 FPOUDIESHGOTDS occaoscccaseesierseresnZe lt peesceethossbiacssusessaeushlucsedetentoaheedscoanctsendyasedsdvesettarsuhbeisdpenshtosdbabhdedveassdusebhsetscssacheunsns EE E EREE A 6 Additional Information soso ce s de ceeesteeteess icdeosecesccesseducseaccentoohbyisesadecenevvabedeaveassenebenseisapedsneoednebhdessensdeesdahsed sesedoesuesuasndscoasysncsaheeds 7 Related Thermo Scientific Products iscriere cirios eE Sire E EEEE E E EEE ENEE EE CEE EE aE SEEEN EE EEE E REEE S 7 Introduction The Thermo Scientific Pierce Direct Magnetic IP Co IP Kit enables efficient antigen immunoprecipitations IP and co immunoprecipitations co IP The activated magnetic beads contain N hydroxysuccinimide NHS functional groups which react with primary amines on antibodies to form stable amide linkages The antibody is first coupled to the NHS activated magnetic beads in an amine free buffer The antibody coupled beads are washed to remove any non covalently bound antibody and quenched The prepared beads are incubated with the antigen sample washed to remove non bound material and eluted in a low pH elution buffer to dissociate the antigen The k
16. ydrophobic Dissolve protein in coupling buffer containing up to coupling buffer 20 DMSO Magnetic beads Magnetic beads were frozen or Handle the beads as directed in the instructions aggregate centrifuged Beads aggregate during Proteins on bead surface adhered to After quenching add 0 05 detergent e g Tween the coupling process tube plastic 20 Detergent to the washes Beads were not thoroughly mixed before and or during incubation Note Do not use detergent in the coupling step Continued on the next page 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 Continued from the previous page Thermo S CTPENTI Antibody detected with the eluted antigen Non bound antibody was not sufficiently removed with the washes following the coupling step Step 5 Increase the number of Elution Buffer washes after coupling The antibody coupled beads were treated with a reducing agent e g DTT during the IP or elution steps which reduced the antibody and eluted antibody fragments or subunits that were not covalently linked to the beads Use buffers that do not contain reducing agents Protein does not elute Elution conditions were too mild Increase incubation time with elution buffer to 10 minutes or use a more stringent elution buffer Component in IP Lysis Wash Buffer interfered with antibody ant
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