Pierce™ Magnetic HA-Tag IP/Co-IP Kit
Contents
1. Plate Plate Name Content Volume Time Speed Anti HA Magnetic Beads 25uL 1 Beads 5 seconds IP Lysis Wash Buffer 175uL 2 Bead Wash IP Lysis Wash Buffer 1000uL 1 minute slow 3 Bind Sample containing HA tagged Protein 300uL 30 minutes slow 4 Wash 1 IP Lysis Wash Buffer 300uL 30 seconds slow 5 Wash 2 IP Lysis Wash Buffer 300uL 30 seconds slow 6 Wash 3 Ultrapure Water 300uL 30 seconds slow 7 Elution Elution Buffer 100uL 10 minutes slow 8 Tip Plate KingFisher 96 ee for Deep Well 10 seconds fast Notes 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 Iflow pH Elution Buffer is selected for elution neutralize the pH by adding 15uL of Neutralization Buffer for each 100uL of eluate upon run completion e If using SDS PAGE Sample Buffer in a heated elution install the KingFisher Flex Heating Block see manual for proper installation to heat samples at 95 100 C for 5 10 minutes Select the protocol using the arrow keys on the instrument keypad and press Start See the KingFisher Flex Instrument User Manual for detailed information Slide open the door of the instrument s protective cover Load plates into the instrument according to the protocol request placing each plate in the2. HA Antibody 26184 Pierce HA Peptide 20290 DTT Dithiothreitol 78260 B PER II Bacterial Protein Extraction Reagent Tween is a trademark of Croda International PLC 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 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 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
3. optimal for the protein protein interaction Insufficient sample loaded on the gel to detect by Western blot Magnetic beads were frozen or centrifuged Buffer was incompatible with magnetic beads Detergent was not added to the wash and bind solutions Elute sample in 30 acetonitrile 0 5 formic acid then Optimize the co IP buffer dry down using a Thermo Scientific Speedvac Vacuum Concentrator Bring the sample back up in SDS PAGE Sample Buffer and load entire elution fraction on to gel Handle the beads as directed in the instructions Additional Information Available on Our Website e Frequently Asked Questions search FAQ e Tech Tip 43 Protein stability and storage search TRO043 on www thermoscientific com pierce e Visit www thermoscientific com kingfisher for information on the KingFisher Products e Inthe U S A purchase KingFisher Supplies from VWR Contact your local Thermo Fisher Scientific office to purchase KingFisher Supplies outside the U S A Pierce Biotechnology PO Box 117 3747 N Meridian Road 6 Rockford IL 61105 USA 815 968 0747 815 968 7316 fax www thermoscientific com pierce Frequently Asked Questions for the KingFisher Instrument Question Answer Which plates are compatible with The KingFisher Flex Instrument is compatible with the KingFisher 24 Deep KingFisher Flex and KingFisher 96 Well Plates Microtiter Deep Well 96 Plates and
4. same orientation Confirm each action by pressing Start 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 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 Troubleshooting Problem Possible Cause Solution Little or no HA tagged protein is detected Tagged protein degraded Include protease inhibitors e g Halt Protease Inhibitor Single Use Cocktail EDTA free Product No 78425 in the Lysis Buffer Use new lysate or lysate stored at 80 C No or minimal tagged protein was expressed Verify protein expression by SDS PAGE or Western blot analysis of the lysate using HA tagged Positive Control as a reference Increase the amount of lysate used for IP co IP Use a more sensitive detection system such as Thermo Scientific SuperSignal West Femto Chemiluminescent Substrate Product No 34095 Failure to co IP interacting protein aggregated Wash conditions were too stringent for the weak or transient interaction Reduce the number of washes and or lower the ionic strength of wash buffer Interacting protein was expressed at a low level Apply additional protein sample Use a more sensitive detection system Magnetic beads Buffer system was not
5. INSTRUCTIONS Thermo Pierce Magnetic HA Tag IP Co IP Kit 88838 2516 0 Number Description 88838 Pierce Magnetic HA Tag IP Co IP Kit contains sufficient reagents to perform 40 reactions using 25uL of magnetic beads Kit Contents HA tagged Positive Control 26180X 500uL 1mg mL Escherichia coli extract containing HA tagged GST PI3K SH2 domain Application Set 88838X Pierce Anti HA Magnetic Beads 1mL supplied at 10mg mL in PBS containing 0 05 Tween 20 Detergent and 0 05 sodium azide Pierce IP Lysis Wash Buffer 2 x 50mL pH 7 4 0 025M Tris 0 15M NaCl 0 001M EDTA 1 NP40 5 glycerol Lane Marker Sample Buffer Non reducing 5X 5mL pH 6 8 0 3M Tris HCl 5 SDS 50 glycerol lane marker tracking dye Elution Buffer 5mL pH 2 0 Neutralization Buffer 1mL pH 8 5 Storage Upon receipt store at 4 C Product shipped with an ice pack Table of Contents TPO GUC HOM eea cadens cetacean tetestescaeececaccensceceuvecanaceticneatecevacscassceiancesuncecvisceucecessaeaeaes taccarscesadnsaseccecdeceusctetaneszecesntecedectsatacisaenestcace 2 Important Product Inform at OM sicer s Ra aE E R EE EO EEE A AE ERE nA scat EEE REARS 2 Additional Materials R guiredisssssinosiriciecsicnorsr nienean anae Aee Ee eA ES AT E VAPE ER AEE Eea Ee aE eei iea Ee Ne EREE EEAO rede 2 Procedure for Lysis of Mammalian Cells cece ceccscecseeeeeeeeeseceeceseceseceaecsaecsaecaeecaeecaeseaeseeeeseseseeaessaessaecsaecsaesaeesseseneeeee
6. KingFisher 96 and 96 PCR Instruments Plates Is it possible to concentrate samples Both deep well plates and KingFisher 96 Plates may be used during the same during the run 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 heating block is located inside the instrument and can be used automatically the run 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 Eluted proteins and proteins conjugated to beads can nonspecifically bind to tips and wells or the eluted proteins plastics Adding detergent to the Binding Wash Buffer prevents the protein stick to the wells conjugated to the bead from sticking e g 0 05 0 1 Tween 20 Detergent Also 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 For best results keep the specified volumes within defined limits to avoid critical spillover Related Thermo Scientific Products 88836 7 Pierce Anti HA Magnetic Beads 88832 3 HisPur Ni NTA Magnetic Beads 88821 2 Pierce Glutathione Magnetic Beads 26180 Pierce HA Tag IP Co IP Kit 26181 2 Pierce Anti HA Agarose 26183 Anti
7. 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 For a faxed copy call 800 874 3723 or contact your local distributor 2012 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
8. e Non reducing Sample Buffer by mixing 200uL of 5X Lane Marker Sample Buffer with 800uL of ultrapure water The 1X solution may be stored at 4 C for up to one year 2 Add 100uL of 1X Non reducing Sample Buffer to the beads Vortex briefly to resuspend the beads and then incubate in a heat block at 95 100 C for 5 10 minutes Note Using Non reducing Sample Buffer can minimize interference from co eluting antibody fragments 3 For reducing gel analysis add 2 5uL of 2M DTT to the 100uL sample 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 Thermo SC MEIN Pre C Automated IP Co IP Note The following protocol is designed for use with the KingFisher Flex Instrument The protocol can be modified according to your needs using the Thermo Scientific BindIt Software provided with the instrument 1 Download the HA_Tag_Immunoprecipitation protocol from the Thermo Scientific website http www thermoscientific com bindit protocols into the BindIt Software on an external computer Transfer the protocol to the KingFisher Flex 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 IP protocol using the Thermo Scientific Microtiter Deep Well 96 Plates
9. eansegs 3 Procedure for IP of HA Tas ged Proteis ccccsccesvesiieiseesececoessesesnedsstervenstdncssescdesscnsbacdseseesenncebncedvecsetuopeubbacspedssderstnnndessensetervenreas 4 Bos IMiantial TR CO IP is acces vnsnd besten EE A O AE EE E EE ecstacy EE AE E ET 4 B Elutionot HA Tagged ProteiM sirisser rinser ea Vre CEE EEEE eE EEE ESE EO ES EER ESE AE TOES VEEE NE aE PNE ESS iea VEEE WEE 4 Cz Automated P COAP oiiire or respei aee ee EE eE rE SEPE EEE EEEE EE EE EET S EEE EEE EEEE E EEEE EE EEPE SEE EE EEEE EE E S 5 Troubleshoot Tss irere esient aene eE E E E E EE EEE ESEE OE EE EEEE Aa EEVEE ESS EE OEE EEEE EEE EESE AE E 6 Additional Information Available on Our Website ecceesceceseceessecesecesseeceeneeenaeceseeessaeceneesnaeceseeessaeceeneesaaeceeeessaeceeneeeaeeeneess 6 Frequently Asked Questions for the KingFisher Instrument ceeccessseseseeceeeceeececeseceeeeecaeceeeecsaeceeneeceaeeeneecaaeceeneeseaeeeeeees 7 Related Thermo Scientitic Products sic scc 555ci sacs cases easncedsenesdec vabeesinedsteecvesedesoesscdennnebuacdoucesdensuabuaeasensedneyeabbasipcdelenrsdaredetieascderoenrbay 7 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 Introduction The Thermo Scientific Pierce Magnetic HA Tag IP Co IP Kit provides a simple and fast method to study HA tagged proteins with advantages over the tradit
10. ion protein mg of beads Important Product Information e Do not centrifuge dry or freeze the Pierce Magnetic Beads Centrifuging drying or freezing will cause the beads to aggregate and lose binding activity e For best results determine optimal conditions for expression of HA tagged fusion protein before attempting IP e Include a non transfected lysate as a negative control to identify nonspecific binding of proteins to the anti HA magnetic beads The HA tagged Positive Control assists in verifying whether the anti HA magnetic beads can successfully capture the HA tagged protein e Cell lysate with expressed HA tagged protein may be prepared from mammalian cells using the procedure provided or from bacterial cells using a bacterial lysis protocol e g Thermo Scientific B PER II Bacterial Protein Extraction Reagent Product No 78260 The buffers provided in this kit are compatible with samples prepared from bacterial lysate e 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 e Binding capacity and elution recovery will vary depending on the HA fusion protein and the method of elution e 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 The HA antibody will
11. ional immunoprecipitation IP procedure using Protein A G magnetic beads The high affinity anti HA antibody coupled beads enable IP of HA tagged proteins or co immunoprecipitation co IP of their interacting partners without antibody contamination The Thermo Scientific Pierce Anti HA Magnetic Beads are used for the IP of specific HA tagged proteins expressed in human in vitro expression systems and mammalian and bacterial cell lysates The anti HA antibody coupled to the blocked magnetic beads is a high affinity mouse IgG monoclonal antibody that recognizes the HA epitope tag YPYDVPDYA derived from the human influenza hemagglutinin HA protein For immunoprecipitation the beads are added to a sample containing HA tagged proteins The bound HA tagged proteins are dissociated from the beads using a low pH elution buffer and removed from the solution manually using a magnetic stand or by automation using an instrument such as the Thermo Scientific KingFisher Flex or KingFisher Duo Instrument Automated instruments are especially useful for large scale screening of multiple samples Table 1 Characteristics of the Thermo Scientific Pierce Anti HA Magnetic Beads Composition High affinity mouse IgG monoclonal antibody covalently coupled to a blocked magnetic bead surface Magnetization Superparamagnetic no magnetic memory Mean Diameter 1um nominal Density 2 0g cm Bead Concentration 10mg mL Binding Capacity gt 10ug GST ERK HA 70kDa fus
12. not leach from the beads when eluting with IgG Elution Buffer pH 2 0 e Pierce Anti HA Magnetic Beads are compatible with IP and analyses by Western blot e Do not use cell lysate containing dithiothreitol DTT DTT may cause the HA antibody to leach from the beads Additional Materials Required e Phosphate buffered saline PBS 100mM sodium phosphate 100mM NaCl pH 7 2 Product No 28372 e DTT Product No 20290 e Sample containing HA tagged protein 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 For Automated IP e KingFisher Flex System with 96 deep well head Product No 5400630 e Thermo Scientific 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 For Manual IP e 1 5mL microcentrifuge tubes e Magnetic stand e g Thermo Scientific MagnaBind Magnet for 6 x 1 5mL microcentrifuge tubes Product No 21359 e End over end rocker or rotator Procedure for Lysis of Mammalian Cells Note For optimal results use a protease inhibitor cocktail such as Halt Protease Inhibitor Cocktail Product No 87786 when preparing cell lysate Protocol I Lysis of Cell Monolayer Adherent Mammalian Cells 1 Carefully remove culture medium from confluent cells 2 Wash the cells once with ice cold PBS 3 Add the ap
13. propriate volume of IP Lysis Wash Buffer Table 2 to the cells Incubate on ice for 5 minutes with periodic mixing 4 Transfer the lysate to a microcentrifuge tube and centrifuge at 13 000 x g for 10 minutes to pellet the cell debris 5 Transfer supernatant to a new tube for protein concentration determination e g Thermo Scientific BCA Protein Assay Kit Product No 23225 and further analysis Table 2 Recommended 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 Protocol II Lysis of Cell Suspension Cultures 1 Centrifuge the cell suspension at 1000 x g for 5 minutes to pellet the cells Discard the supernatant 2 Wash cells once by suspending the cell pellet in PBS Centrifuge at 1000 x g for 5 minutes to pellet cells 3 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 of IP Lysis Wash Buffer to the cell suspension 4 Incubate lysate on ice for 5 minutes with periodic mixing Remove cell debris by centrifugation at 13 000 x g for 10 minutes 5 Transfer supernatant to a new tube fo
14. r protein concentration determination e g BCA Protein Assay Kit Product No 23225 and further analysis 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 Procedure for IP of HA Tagged Proteins A Manual IP Co IP Note The amount of lysate needed and incubation time are dependent upon the expression level of the HA tagged protein and require optimization for each specific system For co IP experiments the buffer system must be optimized to maintain the protein protein interaction Note To ensure homogeneity mix the beads thoroughly before use by repeated inversion gentle vortexing or using a rotation platform 1 Place 25uL 0 25mg of Pierce Anti HA Magnetic Beads into a 1 5mL microcentrifuge tube 2 Add 175uL of IP Lysis Wash Buffer to the beads and gently vortex to mix 3 Place the tube into a magnetic stand to collect the beads against the side of the tube Remove and discard the supernatant 4 Add ImL of IP Lysis Wash Buffer to the tube Invert the tube several times or gently vortex to mix for minute Collect beads with a magnetic stand Remove and discard the supernatant 5 Add the sample containing the HA tagged protein to the pre washed magnetic beads and incubate at room temperature for 30 minutes with mixing Note To prepare the positive control dilute 30uL into 270uL of IP Lysis Wash Buffer 6 Collect the beads
15. with a magnetic stand remove the unbound sample and save for analysis 7 Add 300uL of IP Lysis Wash Buffer to the tube and gently mix Collect the beads and discard the supernatant Repeat this wash twice 8 Add 300uL of ultrapure water to the tube and gently mix Collect the beads on a magnetic stand and discard the supernatant B Elution of HA Tagged Protein Note If the eluted HA tagged protein will be used for functional applications use Elution Protocol 1 to elute the protein If the protein is sensitive to the low pH use Thermo Scientific Pierce HA Peptide Product No 26184 to competitively elute the HA tagged protein Elute in 100uL of Pierce HA Peptide 2mg mL in Tris buffered saline containing 0 05 Tween 20 Detergent for 5 10 minutes at 37 C For electrophoretic analysis use Elution Protocol 2 e Elution Protocol 1 1 Add 100uL of Elution Buffer to the beads Incubate the beads at room temperature with mixing for 5 10 minutes 2 Separate the beads on a magnetic stand and save the supernatant containing the target antigen To neutralize the low pH add 15uL of Neutralization Buffer for each 100uL of eluate 3 For reducing gel analysis prepare Reducing Sample Buffer by adding 2 5uL of 2M DTT and 20uL of Lane Marker Non reducing Sample Buffer 5X 4 Add 77 5uL of elution sample to the 22 5uL of prepared Reducing Sample Buffer Heat sample at 95 100 C for 5 10 minutes in a heat block e Elution Protocol 2 1 Prepar
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