COIPP - Bulletin - Sigma
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1. i 7 Chl c Bai S m By ii ES 5 Sal fi Fi cu i FLAG Bum HI cy 2 pFLAD CIA Di 2 an pole cM p mu a7 b A LA LLL LS 4 LI d A mz 164 A _ HT VUA EXIT dl Ime E eod J al k Clone gene X in THE Expresion Vector lk Clone gene Y in po Myc MV 2 Expression Vector 2 Co express plasmids mammalian cellis 3 Lyse cells o ur 4 Incubabe wiih Anti FI AGS 2 High Senativity Caphare Plate Elta wrth 2 Sample Batter 41 Wash Western blotting Analysis Date 6 Version SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 5 0 IMMUNOPRECIPITATION VECTOR KIT PRODUCT COIP P This kit includes the transient mammalian expression vectors pFLAG CMV 2 and pc Myc CMV 2 which are designed for the construction and expression of N terminally tagged fusion proteins with a FLAG epitope DYKDDDDK or a c Myc epitope EQKLISEEDLY respectively The control plasmids pFLAG CMV 2 p53 pc Myc CMV 2 Large T antigen and pc Myc CMV 2 BAP are also supplied The control plasmids can be used as positive and negative binding partners in the immunoprecipitation analysis see Section 5 4 Figures 2 through 4 and Table illustrate the features of the supplied vectors 5 1 Rea2. 1974 diluted with 150 ul Lysis Buffer Product L1413 to a single well This control sample should be treated identically to all other samples Wash each well on the plate 4 times with 200 ul of 1x Wash Buffer The plate can be washed either manually or with a plate washer After washing remove excess liquid by firmly tapping the plate onto a paper towel Note After this step the captured proteins can be analyzed by two methods ELISA Section 8 5 1 or Western blotting Section 8 5 2 Detection of the Bound Protein by ELISA or Western Blotting After the FLAG and c Myc tagged protein complexes have been captured Section 8 4 they can be detected using either ELISA or Western blotting 8 5 1 1 ELISA Dilute Monoclonal Anti c Myc Clone 9E10 Alkaline Phosphatase Conjugate Product 5963 1 100 with Blocking Buffer Tris Buffered Saline pH 8 0 with 3 Nonfat Milk Add 200 ul of the diluted antibody to each well Cover the plate with the lid to minimize evaporation Incubate the plate for 1 hour at room temperature on a rotary shaker with gentle agitation During the incubation prepare the pNPP substrate solution provided in the Detection Kit and a 3 N NaOH Stopping Solution a Prepare enough pNPP solution for each well being used in the assay To dissolve 1 tablet set add 5 ml of MilliQ water to a sterile 50 ml conical tube and add one SIGMA FAST p Nitrophenyl Phosphate Tablet set one buffer tablet and one pNP
3. Large T antigen expressed in COS 7 cells To ensure that the system works in your hands we recommend including the control plasmids provided in the Immunoprecipitation Vector Kit in your experiment 8 3 Preparation of Cell Extract Aspirate growth medium from the transfected cells to be assayed Rinse the cells twice with ice cold PBS 2 Add 1 ml of ice cold Lysis Buffer supplemented with mammalian protease inhibitors 10 ul per 1 ml Lysis Buffer Product P8340 to a 10 cm plate you can adjust the volume of the Lysis Buffer according to the size of the culture plate used 3 Scrape the cells off the plate with cell scrapers Product C2802 and transfer them to microcentrifuge tubes 4 Incubate for 1 hour on ice occasionally mixing with a vortexer Microcentrifuge the cells for 15 minutes at 4 and transfer the supernatant to a new tube If the supernatant is not to be used immediately store the cell lysate at 70 C Note The supernatant may be viscous during transfer due to the genomic DNA released from the nuclei of lysed cells Genomic DNA be removed from the supernatant with a 1 ml pipet tip prior to the transfer 6 Determine the total protein concentration of the supernatant using standard techniques We recommend using Sigma s BCA Protein Assay Bicinchoninic Acid Kit Cat No BCA 1 84 Immunoprecipitation with ANTI FLAG M2 plate Note For protein complexes where the interaction occurs at salt and deter
4. Note The only difference between the two expression vectors is the epitope tags Date 8 Version SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual Figure 3 Nucleotide sequences of the epitope tags and multiple cloning region of the pFLAG CMV 2 upper and pc Myc CMV 2 lower expression vectors Sequence Range 020 bo 1022 Damo Misi IL ex 1 E Cis l ATS AMA GAT GAC GUI UDC AAT TCA TOT AT TIT Cum CGL AGT PLAG Gring Sauce Kea Zw Aba BaH Seal TOE CTA CDC GAC GEA OGG G GAC SSF Oe CEG AGA COT Goo rnm Sequence Pange 929 bo 10021 Trial s rad kitam Meil il Erak I Cir ct ATS ATC TCA CTO CTT 000 03 TOU TAC CTT GTT TIT GA GAA OG ACT z hipr Codng Em mI Abe Pest 2 41 GAT TCG OCA GAC TOT AGA GGA CE Uu SEF OAG OTG COT Ah GOC T Version 9 Date SIGMA ALDRICH FLAG
5. ANTI FLAG M2 plate is designed to capture recombinant FLAG fusion proteins obtained from cell lysates of transfected cells 6 1 Reagents Provided sufficient for 96 samples ANTI FLAG M2 High Sensitivity P2983 1 blate Capture Plate 20 mM Tris HCl 7 4 1 M NaCl 1 mM DTT 1 0 Triton X 100 oe n B2556 6 ml Amino terminal FLAG BAP Fusion 1974 2 5 ug Protein 6 2 Additional Reagents and Equipment Not Supplied Lysis Buffer 10x Wash Buffer 500 mM Tris HCl pH 7 4 1 5 M NaCl 125 mM Tris HCl pH 6 8 4 SDS 20 v v glycerol 0 004 bromphenol blue 2x Sample Buffer SealPlate Film Pipets single or multi channel Tissue culture incubator Pipette tips Mammalian protease inhibitor cocktail Microcentrifuge tubes Ice cold PBS Reagent reservoir Cell scrapers Microcentrifuge at 4 C Kit for measuring total protein Vortexer concentration MilliQ or sterile water 5 4 C incubator Tissue culture plates Plate washer Tissue culture medium Rotary shaker Tissue culture cells such as COS 7 Date 12 Version SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 7 0 IMMUNOPRECIPITATION DETECTION KIT PRODUCT COIP D The Immunoprecipitation Detection Kit provides all of the unique reagents required to detect protein protein interactions using a plate based colorimetric assay The substrate p Nitrophenyl Phosphate Disodium pNPP is hydrolyz
6. Buffer 6 ml SealPlate Film 6 User Manual Storage Conditions Store all components at 4 C Immunoprecipitation Detection Kit Product COIP D Monoclonal Anti c Myc Clone 9E10 Alkaline Phosphatase Conjugate 0 25 ml Tris Buffered Saline pH 8 0 with 3 Nonfat Milk 1 packet SIGMA FAST p Nitrophenyl Phosphate Tablets 5 tablet sets Technical Bulletins Storage Conditions Store all components at 4 C Date 4 Version SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 3 0 INTRODUCTION Researchers studying protein protein interactions commonly use one of several different two hybrid systems While these systems are of some use in determining protein binding partners all of the current two hybrid systems generate false positives Sigma s FLAG 96 well Immunoprecipitation Kit provides a unique method to validate protein protein interactions in a convenient 96 well format This system enables potential binding partners for the protein of interest to be tagged with FLAG and c Myc epitopes followed by capture and detection using the ANTI FLAG M2 High Sensitivity Capture Plate The system offers the following advantages compared to currently available techniques 1 Rapid 96 well format The 96 well plate configuration is very effective at handling multiple samples and greatly increases the throughput of the assay Additionally this is an ideal system for optimizing in vitro bindi
7. WITH THE ANTI FLAG M2 PLATE 8 1 Overview of the Analysis of Protein Protein Interaction with the ANTI FLAG 2 Plate by ELISA or Western blotting Day 1 Plate cells Day 2 Transfect cells Day 3 or 4 Harvest cells with Lysis Buffer supplemented with mammalian protease inhibitor cocktail and prepare cell lysate Incubate cell lysate with the ANTI FLAG M2 plate for 1 hour at 4 C diluting the cell lysate is optional Wash the plate 4 times with 200 ul per well of the 1 Wash Buffer Analysis with ELISA Add 200 ul of Anti c Myc Alkaline Phosphatase Conjugate diluted 1 100 in Blocking Buffer to each well and incubate for 1 hour at room temperature on a rotary shaker Wash the plate 4 times with 200 ul per well 1x Wash Buffer Incubate each well with 200 ul pNPP substrate Incubate at room temperature for 15 to 30 min or until the yellow color develops Stop the reaction with 3 N NaOH Measure with a plate reader After this step the plate can be analyzed by ELISA or Westem blotting Analysis with Western Blotting Add 60 ul of 2x Sample Buffer supplemented with 10 B Mercaptoethanol to each well and incubate at room temperature for 30 minutes with shaking on a vortexer Transfer the contents of each well to microcentrifuge tube and boil for 5 minutes Western Blotting Date 14 Version SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 8 2 Gene
8. of Forward primer 20 uM Verification Primer MF C 1 ul of Reverse primer 20 Verification Primer MR d 8 ul of Water Note If gene specific primers are used the PCR reaction we recommend that you make a stock concentration of 20 uM and use 1 yl of this stock per PCR 5 Mix gently and briefly centrifuge each sample to collect all of the components at the bottom of the tube 6 Add 25 yl of the PCR master mix prepared in Step 4 into the PCR tubes or the multiwell PCR plate containing 5 ul of the colony mix Date 22 Version SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 11 12 Start the PCR using a thermal cycler with a heated lid The amplification parameters should be optimized for each unique combination of primers template and thermal cycler If the vector primers are used the cycling parameters should be a Incubate at 94 for 5 minutes b 25 cycles of 94 for 40 seconds 55 for 50 seconds and 72 for 2 minutes C Extend at 72 C for 10 minutes Note The proposed extension time is suitable for the amplification of PCR products 0 2 to 2 kb in length You can alter the extension time in proportion to the length of the expected PCR fragments generally 1 kb minute When the PCR is complete analyze 15 yl of the amplified DNA from each PCR by electrophoresis in an agarose EtBr gel When positive PCR products are identified match them to the original PCR tu
9. primer This method allows the identification of recombinant clones with the right orientation In addition to your experimental sample we recommend that you perform a positive control PCR using one of the control plasmids as the template in conjunction with the vector primers This control reaction will confirm that the PCR was successful If control plasmids and the vector primers are used for the positive control PCR the expected PCR fragments are shown in the table below Sizes of Expected PCR fragment kb pFLAG CMV 2 p53 Control Plasmid P9986 1 3 kb Product pc Myc CMV 2 Large T antigen Control Plasmid P9861 2 4 kb pc Myc CMV 2 BAP Control Plasmid P9736 1 7 kb Procedure for Screening Recombinant Plasmids 1 Pick single colonies from the LB Ampicillin agar plate using a sterile pipet tip 2 Deposit the bacteria at the bottom of individual PCR tubes or the wells of a multiwell PCR plates Product 237 490 3 containing 20 ul of water Product W1754 and vortex 3 Aliquot 5 ul of the colony mixture into separate PCR tubes or a multiwell PCR plate and set it aside The rest of the mixture can be left at room temperature until the PCR is completed 4 Prepare a PCR master mix with Sigma s ReadyMix Product P4600 Prepare enough PCR master mix for all of the PCR tubes For each PCR 30 ul total volume mix the following reagents in a sterile microcentrifuge tube a 15 ul of 2x PCR ReadyMix Taq b 1 ul
10. 2 Control Plasmid bal Control Plasmid mad 6 8 kb hGH poly A 6 1 kb hGH poly A 3801 3096 SV40 ori SV40 ori E coli ori b lact 5067 E coli ori b lact 4362 Date 10 Version SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 5 5 Verification Primers Verification primer MF and Verification primer MR are supplied with this kit and allow the researcher to screen for the presence orientation and reading frame of the DNA insert by colony PCR or DNA sequencing Table Il Table Il Vector primers and sequences Verification Primer MF P2987 5 AAT GTC ATA ACC CGT TGA CGC 3 Verification Primer MR P3112 5 TAT TAG GAC AAG GCT GGT GGG CAC 3 Notes 1 Please see Appendix 1 for procedures involved in cloning recombinant plasmids 2 Please see Appendix 2 for procedures involved in screening recombinant plasmids by Colony PCR 3 If the genes of interest are already cloned into expression vectors that contain FLAG or c Myc tags it is not necessary to subclone them into the expression vectors provided in this kit Version 11 Date SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 6 0 FLAG 96 WELL IMMUNOPRECIPITATION KIT PRODUCT HT COIP 1 The FLAG 96 well Immunoprecipitation Kit provides all of the unique reagents required to study protein protein interactions using a 96 well ANTI FLAG M2 High Sensitivity Capture Plate ANTI FLAG M2 plate The
11. 96 well Immunoprecipitation System User Manual 5 4 Control Vectors The Immunoprecipitation Vector Kit provides positive and negative control vectors for co immunoprecipitation Figure 4 prLAG CMV M 2 p53 Control Plasmid expresses the N terminally tagged FLAG fusion protein containing the wild type C terminal domain of mouse p53 residues 73 390 pc Myc CMV 2 Large T antigen Control Plasmid expresses the N terminally tagged c Myc fusion protein containing the C terminal domain of Large T antigen residues 86 708 pc Myc CMV 2 BAP Control Plasmid expresses the N terminally tagged c Myc fusion protein containing bacterial alkaline phosphatase BAP Mammalian cells co transfected with control plasmids 2 p53 and pc Myc CMV 2 Large T antigen will express tagged fusion proteins that interact providing a positive control for the co immunoprecipitation analysis Cells co transfected with pFLAG CMV 2 p53 and pc Myc CMV 2 BAP express tagged fusion proteins that do not interact demonstrating a negative result in the co immunoprecipitation analysis Figure 4 Circular maps of control vectors 5197 166 Hind III p53 coding sequence pFLAG CMV 2 p53 Xba I Control Plasmid 5 6 kb hGH poly A 2612 5 40 ori E coli ori b lact 2631 6838 6133 Xba I Hind III Large T BAP antigen coding coding sequence c Myc sequence c Myc pc Myc CMV 2 Large T antigen
12. P substrate may have degraded Too much cell lysate added to the ANTI FLAG M2 plate Incubation time with pNPP substrate was too long It is unlikely that the background is contributed by nonspecific interactions between the plate and cellular proteins since the ANTI FLAG M2 plate is highly specific It is likely that the pNPP substrate incubation time was too long or the substrate was previously degraded Loss of the 2x Sample Buffer with 10 B mercaptoethanol during the elution step No B mercaptoethanol in the 2x Sample Buffer Solution Add 6 8 M urea and 10 B mercaptoethanol to the 2x Sample Buffer and then mix with the cell lysate Heat at 100 C for 10 minutes before loading on the SDS PAGE gel Check the expression level of the fusion proteins in the co transfected cell lysate by Western blotting using ANTI FLAG M2 antibody to detect the FLAG tagged fusion protein or anti c Myc antibody to detect the c Myc tagged fusion protein If expression level is low optimize the conditions for transfection with the control plasmids Dilute the cell lysate 1 to 5 fold with 1 Wash Buffer e g 1 volume of cell lysate mixed with 1 to 4 volumes of 1x Wash Buffer before immunoprecipitation with ANTI FLAG M2 plate Prepare a fresh stock of pNPP and use it within 1 hour To determine the integrity of the substrate see Appendix 3 Dilute the cell lysate You can do a titration series of the cell lysate to be analyzed and find
13. P tablet Product N1891 to the water b Vortex until the tablets dissolve The pNPP substrate solution should be used within one hour At the end of incubation remove the liquid from each well and wash the wells 4 times with 200 ul each of 1x Wash Buffer Add 200 ul of pNPP substrate to each well Incubate the plate at room temperature for 15 to 30 minutes or until a yellow color develops Note The time required to develop the yellow color will vary for different cell lysates The higher the concentration of fusion proteins in the cell lysate the quicker the yellow color will develop Stop each reaction with 50 ul of the 3 N NaOH Stopping Solution Mix gently by pipetting Note Avoid introducing bubbles into the wells If bubbles are present remove them by gently tapping the plate prior to reading the absorbance Using a spectrophotometer measure the absorbance of each well at 405 nm Version 17 Date SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 8 5 2 Western Blotting 1 oo 12 13 15 16 Add 60 ul of 2x Sample Buffer supplemented with 10 B mercaptoethanol to each sample well on the plate Seal the plate with SealPlate Film Product Z36 965 9 and clamp the plate to a Vortex Genie 2 Mixer or an equivalent device with two bulldog clamps Notes 1 Warm the 2x Sample Buffer Product B2566 at 37 for a few minutes before use 2 Do not cove
14. QUES A brief outline of the recommended cloning technique is supplied below for more information please refer to Sambrook et al 1989 To test the interaction between protein X and Y the gene for protein X is cloned into the prLAG CMV M 2 vector and the gene for protein Y is cloned into the pc Myc CMV 2 vector 1 DNA encoding protein X and protein Y be generated either by PCR amplification or by cutting them out of another plasmid 2 Purify the DNA fragments with a DNA purification kit designed for PCR analysis We recommend using Sigma s GenElute PCR DNA Purification Kit Product GEN PCR 3 Digest both vectors with the appropriate restriction enzyme s dephosphorylate and purify the DNA 4 Ligate the vector and insert 5 Transform the ligation mixtures into competent E coli cells 6 Plate the transformed E coli on LB Ampicillin agar plates T Screen clones using colony PCR or restriction analysis to identify those containing the correct insert Version 21 Date SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual APPENDIX 2 SCREENING OF RECOMBINANT PLASMIDS BY COLONY PCR This procedure describes colony PCR using the vector primers provided in the kit We recommend that you use one gene specific primer and one of the vector primers to perform colony PCR e g a forward gene specific primer and the Verification Primer MR or Verification Primer MF and a reverse gene specific
15. SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual FLAG and ANTI FLAG are registered trademarks of Sigma Aldrich Biotechnology LP The product designations of pFLAG p3XFLAG pFLAG 1 pFLAG 2 pFLAGSHIFT pFLAG CTS pFLAG ATS pFLAG MAC pFLAG CMV YEpFLAG and FLAG BAP are trademarks of Sigma Aldrich Biotechnology LP All other trademarks are the property of their respective owners 2010 Sigma Aldrich Biotechnology LP All rights reserved Date 2 Version SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 1 0 TABLE OF CONTENTS Version 3 Date SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 2 0 SYSTEM COMPONENTS Immunoprecipitation Vector Kit Product COIP P pFLAG CMV 2 Expression Vector 10 ug pc Myc CMV 2 Expression Vector 10 ug pFLAG CMV 2 p53 control plasmid 20 ug pc Myc CMV 2 Large T antigen control plasmid 10 ug pc Myc CMV 2 BAP control plasmid 10 yg Verification Primer MF 2 nmol Verification Primer MR 2 nmol Technical Bulletins Storage Conditions Store all components at 20 C FLAG 96 well Immunoprecipitation Kit Product HT COIP 1 ANTI FLAG M2 High Sensitivity Capture Plate 1 plate Amino terminal FLAG BAP Fusion Protein 2 5 ug 10x Wash Buffer 50 ml Lysis Buffer 50 ml 2x Sample
16. bes or multiwell PCR plates containing the colony mix Take the positive colony mix and use it to inoculate 3 ml of fresh LB medium containing 100 ug ml ampicillin in a 15 ml culture tube Incubate the culture at 37 overnight Isolate plasmid DNA We recommend using Sigma s GenElute plasmid Purification Kit Product PLN 70 Note The vector primers can also be used for DNA sequencing Version 23 Date SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual APPENDIX 3 CAPTURING THE AMINO TERMINAL FLAG BAP FUSION PROTEIN WITH THE ANTI FLAG M2 PLATE AND EVALUATING THE pNPP SUBSTRATE INTEGRITY Dilute the Amino Terminal FLAG BAP Fusion Protein Product A1974 or nontransfected control cell lysate to a concentration of 2 5 ng pl with 1x Wash Buffer 2 To rehydrate the wells add 200 ul of 1x Wash Buffer to each of 6 wells and incubate for 2 minutes 3 Remove the solution from each well and firmly tap the plate on a paper towel until most of the 1x Wash Buffer has been removed 4 Add 200 ul of Amino Terminal FLAG BAP Fusion Protein solution prepared in Step 1 into each well 500 ng well Cover the plate with a lid 6 Incubate the plate at 4 for 1 hour on an orbital shaker operating at 100 rpm Note During the incubation prepare the pNPP substrate solution a Do not prepare the pNPP solution until the plate is incubating as it must be used within 60 minutes b Add 5 ml of Mill
17. ed by Anti c Myc Alkaline Phosphatase conjugated antibody to produce a yellow colored solution that can be read spectrophotometrically at 405 nm Tris Buffered Saline pH 8 0 with 396 Nonfat Milk is used to dilute the Anti c Myc Alkaline Phosphatase Conjugate The pNPP reaction is terminated with 3 NaOH and then read at 405 nm The SIGMA FAST pNPP tablet set provided in the kit produces a ready to use solution after the addition of 5 ml of distilled or deionized water 7 1 Reagents Provided sufficient for 96 samples Kit Components Product Quantity A5963 1x 0 25 ml T8793 1 packet N1891 5 tablet sets 7 2 Additional Reagents and Equipment Not Supplied Pipets single or multi channel P Mercaptoethanol Pipette tips Bulldog clamps Microcentrifuge tubes Boiling water bath Reagent reservoir SDS PAGE gels and apparatus Microcentrifuge at 4 C Protein transfer apparatus Vortexer Protein transfer membrane Plate washer Tris glycine buffer Plate mixer TBST Rotary shaker TBS MilliQ or sterile water Anti mouse IgG peroxidase conjugate 3 N NaOH ANTI FLAG M2 monoclonal antibody 50 ml conical tubes peroxidase HRP conjugate Spectrophotometer capable of i ECL Plus Reagent measuring absorbance at 405 nm Version 13 Date SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 8 0 PROTOCOLS FOR STUDYING PROTEIN PROTEIN INTERACTIONS
18. f the cell lysate is not necessary 4 Negative binding protein partner Inclusion of a negative binding partner along with the FLAG tagged protein construct is critical for verifying the specific protein protein interaction observed Over expression of a fusion protein may cause nonspecific protein protein interactions between the proteins being tested The c Myc BAP vector provided in the Immunoprecipitation Vector Kit can be used as a negative binding protein partner Version 15 Date SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 8 2 4 Detection of endogenous binding partners during Western blotting or ELISA Although this kit and protocol are designed for detecting the c Myc tagged binding partner from co transfected cell lysates using anti c Myc antibody you may use the ANTI FLAG M2 plate to capture endogenous binding partners that interact with the FLAG fusion protein from cells transfected only with the FLAG fusion protein In this situation optimum dilution and incubation times should be determined for the specific antibodies used to detect the endogenous binding partners during Western blotting or ELISA Since the ANTI FLAG M2 plate is coated with monoclonal ANTI FLAG M2 antibody either polyclonal antibody or antibody conjugates should be used for ELISA based detection to avoid problems with cross reactivity Note This protocol has been optimized for analyzing the interaction of FLAG p53 and c Myc
19. gent concentrations different from the 1x Lysis Buffer use the 1x Wash Buffer to dilute the cell lysate If dilution is required first add the required amount of the 1x Wash Buffer to the plate and then add the cell lysate The ANTI FLAG M2 plate is resistant to the following detergents 5 Tween 20 5 Triton X 1 00 0 1 Igepal CA 630 0 1 CHAPS and 0 2 digitonin The ANTI FLAG 2 plate can also be used with 1 0 M NaCl 1 Rehydrate the plate by adding 200 of 1x Wash Buffer to each well 2 Incubate the plate for 2 minutes at room temperature Note If the entire plate is not used at once cover the unused wells firmly with SealFilm to prevent contamination from the used wells 3 Remove the liquid from each well manually or with a plate washer Tap the plate firmly onto paper towel until most of the solution is removed Date 16 Version SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 8 5 Incubate various amounts of cell lysate on the plate using a rotary shaker with gentle rocking for 1 hour at 4 C Cover the plate with the lid Note The ANTI FLAG 2 plate is coated with 200 ul of ANTI FLAG M2 antibody We recommend that the volume of cell lysate added to each well should not exceed 200 ul This kit includes Amino terminal FLAG BAP Fusion Protein as a positive control for immuno precipitation on the plate For a positive control add 0 5 ug of FLAG BAP fusion protein Product
20. gents Provided sufficient for 96 samples o me o pFLAG CMV 2 Expression Vector pc Myc CMV 2 Expression Vector pFLAG CMV 2 p53 Control Plasmid pc Myc CMV 2 Large T antigen Control Plasmid pc Myc CMV 2 BAP Control Plasmid Verification Primer MF Verification Primer MR 5 2 Additional Reagents and Equipment Not Supplied Pipets single or multi channel Thermal cycler Pipette tips PCR tubes or multi well PCR plate Microcentrifuge tubes Tag DNA polymerase Microcentrifuge Restriction enzymes Reagent reservoir Phosphatase suitable for DNA Vortexer Competent E coli cells MilliQ or sterile water LB ampicillin agar plates DNA plasmid purification kit LB medium with 100 ug ml Ampicillin Transfection reagent 15 ml culture tubes Agarose gel electrophoresis system 37 incubator Version 7 Date SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 5 3 Expression Vectors Figure 2 Circular maps of the expression vectors 4679 Hind 4688 Hind Not EcoR Cla Bgl Il Bgl Il EcoR V EcoR V Kpn Kpn Sal Sal Xba Xba Bam HI Bam HI i Smal Smal pFLAG CMVTM 2 2 4 7 kb 4 7 kb hGH poly A hGH poly A 1642 1651 SV40 ori SV40 ori E coli ori b lact 2908 E coli ori b lact 2917 Table 1 Expression vector features pFLAG CMV 2 pc Myc CMV 2 4679 bp 4688 bp Translational initiation
21. iQ water to a sterile 50 ml conical tube C Add one SIGMA FAST p Nitrophenyl Phosphate Tablet set one buffer tablet and one pNPP tablet Product 1891 to the water d Vortex until the tablets dissolve T Remove the solution from each well and firmly tap the plate on a paper towel until most of the solution has been removed 8 Wash each well 4 times with 200 ul of 1x Wash Buffer 9 Remove the solution and tap the plate dry onto a paper towel 10 Add 200 ul of the pNPP solution to each well 11 Incubate at room temperature for 15 minutes 12 Immediately at the end of the incubation add 50 ul of 3 N NaOH to each well keep the pipette tips submerged in the wells 13 Mix the solution several times by pipetting This step stops the color development and distributes the yellow color evenly in each well Note Avoid the formation of bubbles If bubbles form disrupt them with a clean pipette tip 14 Using a spectrophotometer measure the Ayos with a plate reader If the control immuno precipitation worked correctly and the pNPP solution is fresh and prepared properly the reading should be between 0 5 and 0 7 Date 24 Version
22. ng conditions 2 No leaching of ANTI FLAG M2 antibody The ANTI FLAG M2 antibody is covalently coupled to the surface of the well and the elution conditions used are relatively mild so very little antibody leaches from the plate This results in little to no interference from denatured antibody heavy and light chain bands during Western blot analysis 3 Rapid protocol with quantitative results Because an ELISA format can be used with this system the protein protein interaction assay is rapid less than 4 hours sensitive and quantitative Version 5 Date SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 4 0 FLAG 96 WELL IMMUNOPRECIPITATION SYSTEM OVERVIEW Figure 1 Outline of the procedure Clone two potentially interacting proteins into the two mammalian epitope tagged expression vectors provided in the Immunoprecipitation Vector Kit One vector generates an N terminally tagged FLAG fusion protein while the other vector produces an N terminally tagged c Myc fusion protein Co express the proteins in appropriate mammalian host cells Prepare cell extracts with Lysis Buffer Dilute the cell lysate with 1x Wash Buffer if necessary Incubate cell lysate in the 96 well ANTI FLAG M2 High Sensitivity Capture Plate Wash the ANTI FLAG M2 High Sensitivity Capture Plate with 1x Wash Buffer Analyze captured proteins by ELISA or Western blotting 1 d ENS gt
23. r the plate with a plastic lid since this combination may be too thick for the bulldog clamps Shake the plate for 30 minutes at room temperature on the vortexer at a setting of 3 Transfer the entire contents of each well to separate microcentrifuge tubes Boil each sample for 5 minutes Load approximately 50 ul of each sample onto a standard SDS PAGE gel Transfer the proteins from the gel to a membrane such as PVDF using Tris Glycine Buffer 25 mM Tris 192 mM glycine 15 v v methanol pH 8 3 Product T4904 and the settings recommended by the manufacturer of your transfer apparatus Block the membrane with Blocking Buffer at room temperature for 30 to 60 minutes Transfer the membrane to a tray containing a 1 2 000 dilution of ANTI FLAG M2 Monoclonal Antibody Peroxidase Conjugate Product 8592 or a 1 100 dilution in Blocking Buffer of Monoclonal anti c Myc Unconjugated Clone No 9E10 Product M5546 Incubate at room temperature for 1 hour with gentle agitation Wash the membrane three times 5 minutes per wash with TBST at room temperature with gentle agitation For membranes probed with ANTI FLAG M2 Monoclonal Antibody Peroxidase HRP Conjugate develop the membrane with ECL Plus Reagent according to the manufacturer s instructions For membranes probed with Monoclonal anti c Myc Unconjugated Clone No 9E10 incubate the membrane with anti mouse IgG peroxidase conjugate Product A9044 as the seconda
24. ral Notes Please read this section carefully before proceeding with the experiment 8 2 1 Plasmid DNA for transfection To obtain maximum transfection efficiency the DNA used for transfections must be of high quality free of protein RNA and chemical contamination with an A260 A280 ratio of 1 8 1 9 To produce high quality plasmid DNA we recommend Sigma s GeneElute Endo Free Plasmid Purification Kit Product PLEX 15 8 2 2 Transfection reagent The quality of a transfection reagent dictates the sensitivity and specificity of an assay using the FLAG 96 well Immunoprecipitation Kit Generally high transfection efficiency yields a more sensitive assay requiring a smaller sample volume The Immunoprecipitation Vector Kit includes three control plasmids for optimizing and monitoring the efficiency of transfection To obtain high efficiency transfections we recommend Sigma s ESCORTII Transfection Reagent Product L6037 for commonly used cell lines 8 2 3 Cell lysates used for co immunoprecipitation assay 1 Expression level of the fusion proteins in the cell lysate The concentration of the expressed fusion protein is critical to the success of the co immunoprecipitation analysis The expression of the FLAG and c Myc fusion proteins should be verified by Western blotting with ANTI FLAG M2 antibody and anti c Myc antibody prior to the co immunoprecipitation analysis In general a high concentration of fusion protein in the cell lysate
25. ry antibody diluted 1 80 000 in Blocking Buffer Incubate the membrane at room temperature for 30 minutes with gentle agitation Wash the membrane three times 10 minutes per wash with TBST at room temperature with gentle agitation Rinse the membrane once with TBS Product T6664 to remove residual Tween 20 Develop the membrane with ECL Plus Reagent according to the manufacturer s instructions Date 18 Version SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 9 0 TROUBLESHOOTING GUIDE Problem Precipitation of the cell lysate after mixing with 2x Sample Buffer for Western blotting Samples exhibit no signal or low signal in both Western blotting and ELISA after capturing on the ANTI FLAG M2 plate Samples exhibit high signal in ELISA High background during ELISA Low yield of eluted protein for Western blotting analysis after capturing proteins on the ANTI FLAG M2 plate Possible Cause You may experience a precipitation problem when you mix undiluted cell lysate from certain mammalian cell lines with 2x Sample Buffer The Lysis Buffer is very efficient at solubilizing proteins from cells which sometimes results in too much protein in the lysate and causes aggregation The concentration of the fusion protein in the cell lysates may be low High salt and detergent present in the Lysis Buffer may interrupt protein protein interactions In the ELISA analysis the pNP
26. the optimum amount of cell lysate to generate reading between 0 5 and 1 0 Decrease the incubation time with pNPP Decrease the incubation time with the pNPP substrate or prepare fresh substrate To examine the integrity of the substrate please see Appendix 3 Do not vortex the plate too vigorously which may spill the 2x Sample Buffer Sample Buffer with 10 B mercaptoethanol into adjacent wells Set the vortexer at a speed that provides adequate motion for covering the upper portions of each well 10 B mercaptoethanol is critical for efficient elution of bound proteins Add this to your 2x Sample Buffer Version 19 Date SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual 10 0 REFERENCES 1 Hopp T V Prickett K S Price R T Libby R T March C J Cerretti D Urdal D L and Conlon P J Bio Technology 6 1204 1210 1988 Evan G l Lewis G K Ramsay G and Bishop V M Mol Cell Biol 5 3610 3616 1985 Iwabuchi K M Li B Bartel P and Fields S Oncogene 8 1693 1696 1993 Li B and Fields S FASEB J 7 957 963 1993 Sambrook J Fritsch E F and Maniatis T Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY 1989 e dem Date 20 Version SIGMA ALDRICH FLAG 96 well Immunoprecipitation System User Manual APPENDIX 1 CONSTRUCTION OF FUSION GENES USING STANDARD CLONING TECHNI
27. will yield more sensitive results For each tag the control plasmids supplied in the Immunoprecipitation Vector Kit can be used as positive controls in transfection and Western blot analysis FLAG p53 can be detected with ANTI FLAG M2 antibody while the c Myc Large T antigen and c Myc BAP can be detected with monoclonal anti c Myc antibody 9E10 2 Amount of cell lysate used in the co immunoprecipitation assay The optimum amount of cell lysate added to the wells of the ANTI FLAG M2 plate may vary with each experimental system and should be determined empirically The ANTI FLAG M2 plate based immuno precipitation is very sensitive especially in ELISA When analyzing the interaction of FLAG p53 and c Myc Large T antigen the c Myc Large T antigen can be detected in as little as 5 ug of the cell lysate from co transfected COS 7 cells as compared to the negative control cell lysate Note The volume of the cell lysate used should not exceed 200 pl since the wells of the ANTI FLAG M2 plate are coated up to the 200 ul level with the ANTI FLAG M2 antibody 3 Dilution of the cell lysate The relative binding affinity for different protein complexes may vary Since high salt and detergent levels are present in the Lysis Buffer it may be necessary to dilute the cell lysate with 1x Wash Buffer to minimize disruption of the protein protein interactions Note Forthe positive control interaction between FLAG p53 and c Myc Large T antigen dilution o
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