Pierce™ Protein A/G Magnetic Beads
Contents
1. A ne ee te 10 Additional Materials Required 1 5mL microcentrifuge tubes Wash Buffer Tris buffered saline TBS Product No 28360 containing 0 05 Tween 20 Detergent and 0 5M NaCl Low pH Elution Buffer IgG Elution Buffer pH 2 0 Product No 21028 or 0 1M glycine pH 2 0 Alternative Elution Buffer SDS PAGE reducing sample buffer Antibody for immunoprecipitation Antigen Sample Cell Lysis Buffer used to adjust IP reaction volume Neutralization Buffer High ionic strength alkaline buffer such as a 1M phosphate or 1M Tris pH 7 5 9 Magnetic stand e g MagnaBind Magnet for 6 x 1 5mL Microcentrifuge Tubes Product No 21359 Immunoprecipitation Note This protocol is a general guideline for immunoprecipitation and will require optimization for each application Combine the antigen sample with 10ug of antibody Adjust the reaction volume to 500uL with the Cell Lysis Buffer Incubate the reaction for 1 2 hours at room temperature or overnight at 4 C with mixing Place 25uL 0 25mg of Pierce Protein A G Magnetic Beads into a 1 5mL microcentrifuge tube Add 175uL of Wash Buffer to the beads and gently vortex to mix Place the tube into a magnetic stand to collect the beads against the side of the tube Remove and discard the supernatant Add 1mL of Wash Buffer to the tube Invert the tube several times or gently vortex to mix for 1 minute Collect beads with magnetic stand Remove and discard the supernatant Add the antig2. Materials Required 1 5mL microcentrifuge tubes Sample serum concentrated cell culture supernatant or concentrated ascites Note Samples can be concentrated using the Pierce Concentrators 20mL 20K Product No 87751 or 89887A Binding Wash Buffer Tris buffered saline TBS Product No 28360 containing 0 05 Tween 20 Detergent Elution Buffer IgG Elution Buffer pH 2 0 Product No 21028 or 0 1M glycine pH 2 0 Neutralization Buffer High ionic strength alkaline buffer such as a 1M phosphate or 1M Tris pH 7 5 9 Magnetic stand e g Thermo Scientific MagnaBind Magnet for 6 x 1 5mL Microcentrifuge Tubes Product No 21359 Antibody Purification from Serum Cell Culture Supernatant or Ascites Note To ensure homogeneity mix the beads thoroughly before use by repeated inversion gentle vortexing or using a rotating platform Place 50uL 0 50mg of Pierce Protein A G Magnetic beads into a 1 5mL microcentrifuge tube Add 150uL of Binding Wash buffer to the beads and gently vortex to mix Place the tube into a magnetic stand to collect the beads against the side of the tube Remove and discard the supernatant Add ImL of Binding Wash Buffer to the tube Invert the tube several times or gently vortex to mix for 1 minute Collect beads with magnetic stand then remove and discard the supernatant Dilute 10uL of sample with 490uL Binding Wash Buffer Note Sample volume can be modified according to user preference If the sample volu
3. same wells in each plate For example if using wells Al through A12 use these same wells in all plates e To ensure bead homogeneity mix the vial thoroughly by repeated inversion gentle vortexing or rotating platform before adding the beads to plate 1 e Combine the Tip Comb with a Deep Well 96 Plate See KingFisher Flex Instrument user manual for detailed instructions e Sample volume can be modified according to user preference If the sample volume is lt 500uL dilute it to a final volume of 500uL with Binding Wash Buffer 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 MENGE Pare Executing the Antibody Purification Protocol on the KingFisher Flex Select the protocol using the arrows on the instrument keypad and press Start See KingFisher Flex User Manual for detailed information Slide open the door of the instrument s protective cover Load the plates into the KingFisher Flex according to the protocol request placing each plate in the same orientation Confirm each action by pressing Start After sample processing remove plates as instructed by the instrument s display Press Start after removing each plate Press Stop after all plates are removed Upon completion if desired neutralize the low pH by adding 15uL of Neutralization Buffer for each 100uL of eluate Procedure for Manual Immunoprecipitation
4. INSTRUCTIONS Thermo Pierce Protein A G Magnetic Beads 88802 88803 2339 4 Number Description 88802 Pierce Protein A G Magnetic Beads 1mL supplied at 10mg mL in water containing 0 05 NaN 88803 Pierce Protein A G Magnetic Beads 5mL supplied at 10mg mL in water containing 0 05 NaN Storage Upon receipt store at 4 C Product shipped with an ice pack Table of Contents TOUCH OD ese csees casede ce deere sesenesacas des E cede caseetcncascemtedacotenda cede E E E E E EE 1 Important Product Information 5 22 05 ccccsssscsssecossecescacesinecsveccsovisssacsusocesincssoctsivacessgucdigcesececeuccdsccuuestcesnoedsnacsscoevorpecsscssaucessecseusinses 2 Procedure for Manual Antibody Purification eeecceseceesceceseeeeseeceseeeenceceeeecsaeceeeecaaececeeesaeceeeessaeceeeeessaeceeneeesaeceeeeesaeeeeeees 2 Procedure for Automated Antibody Purification cece eeeeceeseeesceseeesecesecssecsaecaaecaeecseseaeesaeeeeeseesecsaecsaecsaecsaesseseaeseaeeeeeeaeeeas 3 Procedure for Manual Immunoprecipitation cece eeeeceseceesceceseeesseeceeeesneecesneesnaeceseeesseeceneeenaeceeeecsaeceeneessueceeeecaeeeeneeenaeeeneees 4 Procedure for Automated Immunoprecipitation 00 0 0 eee eeeeeeeeeceeeceseceseceaeceaecsuecaeecaeecaeeeaeeeeesseeesesaeesaessaecsuecseesseesseseeeeeeeeenaegs 5 TPOUDIESHOOUDG sser reire aeaii a eE a E EEEE EE EEE EEE EEEE EE RA EEEN EPEE iea ER VEERE EE OEE EEEE EE ASE EEEE E 6 Additional Information Available on Our Website ce
5. It Software User Manual for detailed instructions on importing protocols Set up plates according to Table 3 Table 3 Pipetting instructions for the immunoprecipitation protocol using the Microtiter Deep Well 96 Plates Plate Plate Name Content Volume Time Speed Protein A G Beads 25uL 1 Beads n 5 seconds Binding Buffer 175uL 2 Bead Wash Binding Buffer 1000uL 1 minute Slow 3 Bind Anibudy Anugen sjy 1 hour Slow Sample 4 Wash 1 Wash Buffer 500uL 30 seconds Slow 5 Wash 2 Wash Buffer 500uL 30 seconds Slow 6 Wash 3 Ultrapure Water 500uL 30 seconds Slow 7 Elution Elution Buffer 100uL 10 minutes Medium KingFisher Flex 96 8 Tip Plate Tip Comb for Deep 10 seconds Fast Well Magnets 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 Notes Thermo SC PENG Pre e If less 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 To ensure bead homogeneity mix the vial thoroughly by repeated inversion gentle vortexing or rotating platform before adding the beads to Plate 1 e Combine the Tip Comb with a Deep Well 96 Plate See the instrument user manual for detailed instructions e The beads can be eluted into 100uL of 0 1M glycine pH 2 3 or 100uL of SDS PAGE reducing sample buffer If using SDS PAGE reducing sample
6. buffer in a heated elution install the KingFisher Flex Heating Block see manual for proper installation to heat samples at 96 100 C for 10 minutes e If you select SDS PAGE reducing sample buffer for elution and will be performing a Western blot using rabbit antibodies primary or secondary do not heat the samples Incubate at room temperature for 10 minutes e Iflow pH elution buffer is selected for elution neutralize the pH using 15uL Neutralization Buffer for each 100uL of eluate upon run completion e To limit evaporation select Mix and Slow speed under the subheading Heating Action C Executing Automated Immunoprecipitation Protocol 1 Select the protocol using the arrow keys on the instrument keypad and press Start See the KingFisher Flex Instrument User Manual for detailed information 2 Slide open the door of the instrument s protective cover 3 Load plates into the instrument according to the protocol request placing each plate in the same orientation Confirm each action by pressing Start 4 After the samples are processed remove the plates as instructed by the instrument s display Press Start after removing each plate Press Stop after all the plates are removed Troubleshooting Problem Possible Cause Solution Low amount of protein The protein degraded Add protease inhibitors was recovered Not enough magnetic beads were used Increase the amount of magnetic bead used for
7. capture Sample had an insufficient amount of target protein Increase amount of antigen sample Protein does not elute Elution conditions were too mild Increase incubation time with elution buffer or use more stringent elution buffer Bands at 50kDa appeared on the Western blot Elution was performed in Lane Marker Sample Buffer at temperatures above room temperature and rabbit antibody was used in the Western blot detection Perform elution at room temperature when using a rabbit antibody for Western blot detection Multiple nonspecific bands Nonspecific protein bound to the magnetic beads Add 50 350mM of NaCl to the Binding Wash and Elution Buffers Recovered protein was inactive Elution conditions were too stringent Use a milder elution buffer e g Thermo Scientific Gentle Elution Buffer Product No 21034 Magnetic beads aggregated Magnetic beads were frozen or centrifuged Buffer was incompatible with magnetic beads Handle the beads as directed in the instructions PO Box 117 Rockford IL 61105 USA 6 Pierce Biotechnology 3747 N Meridian Road 815 968 0747 815 968 7316 fax www thermoscientific com pierce Additional Information Available on Our Website e Frequently Asked Questions e Tech Tip 43 Protein stability and storage e Tech Tip 34 Binding characteristics for immunoglobulins and Protein L A G and A G e Visit www ther
8. eececeecceescesscesecesecsaeceecaeecaeecaeseaeseaeeeeesesaeesaecsaecsaecaeecseeeseseneeeeeeeseas 7 Frequently Asked Questions for the KingFisher Instrument s sssessseesneeeereesreesreeerereterrstrestrsstrssrretensteneestttrnreesressressresreeereeeet 7 Related Thermo Scientific Products c sic s iecsssctisssssbedeceonecapeoehstarcsasegsncbsasudnascnsvenesonsnis pense cocdeashdedssnscsunsassedssasncnseodend EEVEE 7 Introduction The Thermo Scientific Pierce Magnetic Beads Table 1 provide a fast and convenient method for both manual and automated magnetic isolation of proteins using affinity binding Pierce Protein A G Magnetic Beads are typically used for isolating antibodies from serum cell culture supernatant or ascites and for immunoprecipitation and co immunoprecipitation of antigens from cell or tissue extracts For antibody purification the beads are incubated with the antibody solution and then magnetically separated from the supernatant For immunoprecipitation the beads are added to an antigen containing sample The bound antibodies or antigens are dissociated from the beads using an elution buffer The beads are removed from the solution manually using a magnetic stand or by automation using an instrument such as the Thermo Scientific KingFisher Flex Automated instruments are especially useful for large scale screening of multiple samples The Pierce Protein A G Magnetic Beads contain a recombinant Protein A G 50 500Da apparent mo
9. en sample antibody mixture to a 1 5mL microcentrifuge tube containing pre washed magnetic beads and incubate at room temperature for 1 hour with mixing Collect the beads with a magnetic stand and then remove the flow through and save for analysis Add 500uL of Wash Buffer to the tube and gently mix Collect the beads and discard the supernatant Repeat wash twice Add 500uL of purified water to the tube and gently mix Collect the beads on a magnetic stand and discard the supernatant Low pH Elution Add 100uL of Low pH Elution Buffer to the tube Incubate the tube at room temperature with mixing for 10 minutes Magnetically separate the beads and save the supernatant containing target antigen To neutralize the low pH add 15uL of Neutralization Buffer for each 100uL of eluate Alternative Elution Add 100uL of SDS PAGE reducing sample buffer to the tube and heat the samples at 96 100 C in a heating block for 10 minutes Magnetically separate the beads and save the supernatant containing target antigen Note If you will be performing a Western blot using rabbit antibodies primary or secondary do not heat the samples Incubate at room temperature for 10 minutes with mixing 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 Procedure for Automated Immunoprecipitation A B Additional Materials Required KingFisher Flex with 96 deep we
10. il EDTA free Product No 78425 in preparation of cell lysates A low pH elution may be used for single use applications Optimal time for low pH elution is 10 minutes exceeding 10 minutes may result in nonspecific binding and yield reduction When using rabbit antibodies primary or secondary in downstream Western blot applications perform elution in SDS PAGE sample buffer at room temperature For all other antibody species boiling the beads in SDS PAGE sample buffer is acceptable for single use applications Boiling could cause bead aggregation and loss of binding activity Pierce Protein A G Magnetic Beads are compatible with small scale antibody purification and immunoprecipitation and analyses by Western blot and mass spectrometry Protein A G has a broader binding range than either Protein A or Protein G individually Protein A G binds to all human IgG subclasses binds somewhat to IgA IgE IgM and to a lesser extent IgD Unlike Protein G Protein A G does not bind serum albumin because the gene sequence coding for the albumin binding site has been eliminated Protein A G is effective for mouse monoclonal antibody purification from IgG subclasses because Protein A G binds all mouse IgG subclasses but does not bind murine IgA IgM or serum albumin For more information see Tech Tip 34 Binding Characteristics for Immunoglobulins and Protein L A G and A G from our website Procedure for Manual Antibody Purification A Additional
11. imited 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 Buyer Any model or sample furnished to Buyer is merely illustrative of the general type and quality of goods and does not represent that any Product will conform to such model or sample 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 REPAIR REPLACEMENT OF OR REFUND FOR THE NON CONFORMING PRODUCT S AT SELLER S SOLE OPTION THERE IS NO OBLIGATION TO REPAIR REPLACE OR REFUND FOR PRODUCTS AS THE RESULT OF I ACCIDENT DISASTER OR EVENT OF FORCE MAJEURE II MISUSE FAULT OR NEGLIGENCE OF OR BY BUYER II USE OF THE PRODUCTS IN A MANNER FOR WHICH THEY WERE NOT DESIGNED OR IV IMPROPER STORAGE AND HANDLING OF THE PRODUCTS Unless otherwise expressly stated on the Product or in the documentation accompanying the Product the Product is intended for research only and is not to be used for any other purpose including without limitation unauthorized commercial uses in vitro diagnostic uses ex vivo or in vivo therapeutic uses or any type of consumption by or application to humans or animals Current product instructions are available at www thermoscientific com pierce F
12. lecular weight by SDS PAGE 40 45K that combines the IgG binding domains of both Protein A and Protein G Protein A G contains four Fc binding domains from Protein A and two from Protein G making it a more general and convenient tool for investigating and purifying immunoglobulins Also Protein A G binding to immunoglobulins is not as pH dependant as Protein A Table 1 Characteristics of the Thermo Scientific Pierce Protein A G Magnetic Beads Composition Recombinant Protein A G monolayer covalently coupled to a blocked magnetic bead surface Magnetization Superparamagnetic no magnetic memory Mean Diameter 1 um nominal Density 2 0g cm Bead Concentration 10mg mL Binding Capacity 55 85ug rabbit IgG mg of bead 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 Important Product Information Do not centrifuge dry or freeze the Pierce Magnetic Beads Centrifuging drying or freezing will cause the beads to aggregate and lose binding activity To ensure good dispersal of beads for optimal antibody binding it is important to include 0 05 non ionic e g Tween 20 Detergent or zwitterionic e g CHAPS detergent in the binding buffer and mix the beads during incubation To minimize protein degradation include protease inhibitors e g Thermo Scientific Halt Protease Inhibitor Single Use Cockta
13. ll head Product No 5400630 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 1 5mL microcentrifuge tubes Binding Buffer Tris buffered saline TBS Product No 28360 containing 0 05 Tween 20 Detergent Wash Buffer Tris buffered saline TBS Product No 28360 containing 0 05 Tween 20 Detergent and 0 5M NaCl Low pH Elution Buffer IgG Elution Buffer pH 2 0 Product No 21028 or 0 1M glycine pH 2 0 Alternative Elution Buffer SDS PAGE reducing sample buffer Antibody for immunoprecipitation Antigen Sample Cell Lysis Buffer used to prepare the antigen sample Neutralization Buffer High ionic strength alkaline buffer such as a 1M phosphate or 1M Tris pH 7 5 9 Instrument Preparation and Plate Set up Note The following protocol is designed for general use with the KingFisher Flex Instrument The protocol can be modified according to your needs using the BindIt Software provided with the instrument 1 Combine antigen sample with 2 10ug of immunoprecipitation antibody per sample Incubate 1 2 hours at room temperature or overnight at 4 C with mixing Download the Immunoprecipitation protocol from the Thermo Fisher Scientific website http www thermoscientific com bindit protocols into the BindIt Software on an external computer Transfer the protocol to the KingFisher Flex from an external computer See Bind
14. me is lt 500uL dilute it to a final volume of 500uL with Binding Wash Buffer Add the diluted sample to the tube containing pre washed magnetic beads and gently vortex or invert to mix Incubate the samples at room temperature with mixing for hour Collect the beads with a magnetic stand then remove and discard the supernatant 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 Pre 8 Add 500uL of Binding Wash Buffer to the tube mix well collect the beads with a magnetic stand and discard the supernatant Repeat this wash twice 9 Add 100uL of Elution Buffer to the tube mix well and incubate 10 minutes at room temperature with occasional mixing 10 Collect the beads with a magnetic stand and then remove and save the supernatant that contains the eluted antibody To neutralize the low pH add 10uL of Neutralization Buffer for each 100uL of eluate Note 50uL is the minimum volume of beads recommended for antibody purification Procedure for Automated Antibody Purification A Additional Materials Required e KingFisher Flex with 96 deep well head Product No 5400630 e Microtiter Deep well 96 Plate V bottom polypropylene 100 1000uUL Product No 95040450 e KingFisher Flex 96 Tip Comb for Deep Well Magnets Product No 97002534 e Binding Wash Buffer Tris buffered saline TBS Product No 28360 contai
15. moscientific com kingfisher for information on the KingFisher Products e Inthe U S A purchase KingFisher Supplies from Fisher Scientific Contact your local Thermo Fisher Scientific office to purchase KingFisher Supplies outside the U S A Frequently Asked Questions for the KingFisher Instrument Question Answer Which plates are compatible with KingFisher Flex Instrument The KingFisher Flex Instrument is compatible with the KingFisher 24 Deep Well Plates Microtiter Deep Well 96 Plates KingFisher 96 and 96 PCR Plates Is it possible to concentrate samples during the run Both deep well plates and KingFisher 96 Plates can be used during the same run Therefore it is possible to start the processing using larger volumes in a deep well plate and elute the purified sample to a smaller volume in a KingFisher 96 Plate Is it possible to heat the samples during the run The heating block is located inside the instrument and can be used automatically during the sample process All plates compatible with the KingFisher Flex Instrument can be heated using specially designed interchangeable heating blocks Why do the beads stick to the plastic tips and wells or the eluted protein sticks to the wells Proteins conjugate to beads and eluted proteins can nonspecifically bind to plastics Adding detergent to Binding Wash Buffer prevents the protein conjugated to the bead from sticking 0 05 0 1 Tween 20 Detergent Al
16. ning 0 05 Tween 20 Detergent e Elution Buffer IgG Elution Buffer pH 2 0 Product No 21028 or 0 1M glycine pH 2 0 e Neutralization Buffer High ionic strength alkaline buffer such as a 1M phosphate or 1M Tris pH 7 5 9 B Preparation of Instrument and Plate Set up Note The following protocol is designed for general use with the KingFisher Flex Instrument The protocol can be modified according to customer needs using the Thermo Scientific BindIt Software provided with the instrument 1 Download the Antibody Purification protocol from the Thermo Fisher Scientific website http www thermoscientific com bindit protocols into the BindIt Software on an external computer 2 Transfer the protocol to the KingFisher Flex from an external computer See BindIt Software User Manual for detailed instructions on importing protocols 3 Set up the plates according to Table 2 Table 2 Pipetting instructions for the antibody purification protocol using the Microtiter Deep Well 96 Plates Plate Plate Name Content Volume Protein A G beads 50uL 1 Beads ET Binding Wash Buffer 150uL 2 Bead Wash Binding Wash Buffer 1000uL 3 Bad Sample 10uL Binding Wash Buffer 490uL 4 Wash 1 Binding Wash Buffer 500uL 5 Wash 2 Binding Wash Buffer 500uL 6 Wash 3 Water 500uL 7 Elution Elution Buffer 100uL 8 Tip Plate KingFisher Flex 96 Tip Comb for Deep Well Magnets Notes e Ifusing less than 96 wells fill the
17. or a faxed copy call 800 874 3723 or contact your local distributor 2013 Thermo Fisher Scientific Inc All rights reserved Tween is a trademark of Croda International PLC All other trademarks are the 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
18. so include a small amount of detergent in the elution buffer e g 0 05 Tween 20 Detergent or silanize the elution plate Are the reagent volumes in each well critical For best results keep the specified volumes within defined limits to avoid spillover Related Thermo Scientific Products 88804 Pierce Classic Magnetic IP Co IP Kit 88805 Pierce Crosslink Magnetic IP Co IP Kit 88845 6 Pierce Protein A Magnetic Beads 88847 8 Pierce Protein G Magnetic Beads 88849 50 Pierce Protein L Magnetic Beads 88816 7 Pierce Streptavidin Magnetic Beads 88826 7 Pierce NHS Activated Magnetic Beads 88828 Pierce Direct Magnetic IP Co IP Kit 24615 Imperial Protein Stain 1L 34075 SuperSignal West Dura Extended Duration Substrate 25200 44 Precise Gels see catalog or website for a complete listing PO Box 117 Rockford IL 61105 USA 7 Pierce Biotechnology 3747 N Meridian Road 815 968 0747 815 968 7316 fax www thermoscientific com pierce Products are 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 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 l
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