65 DETECTION AND PURIFICATION

FLAG® A Proven System for Detection and Purification of Proteins The FLAG Expression System is an established way to express, purify and detect recombinant fusion proteins. FLAG and 3xFLAG have proven utility in numerous applications such as Western blotting, immunocytochemistry, immunoprecipitation, flow cytometry, protein purification, and in the study of protein-protein interactions, cell ultrastructure, and protein localization. These small hydrophilic tags facilitate superior detection and purification of recombinant fusion proteins when using our highly specific and sensitive ANTI-FLAG® antibodies. Read more about the FLAG system in the Cloning and Expression Section.

Product Package Number Size Description Characteristics Applications

FLAG Affinity Gels A 4596 1 ml ANTI-FLAG® M1 Specificity: N-terminal FLAG fusion proteins. Binding • Immunoprecipitation 5 ml Agarose Affinity Gel Ca2+-dependent; the complex dissociates in the absence • Purification of 10 ml of calcium ions. Does not bind to Met-FLAG fusion N-terminal FLAG 25 ml proteins, will not recognize unprocessed, cytoplasmically fusion proteins expressed proteins. Binding Capacity: ≥ 0.6 mg protein per ml gel, binding requires Ca2+ Elution: FLAG peptide; glycine, pH 3.5; EDTA Form: Suspension of beaded agarose in 50% glycerol containing 10 mM sodium phosphate, 150 mM NaCl,

pH 7.4, 0.02% (w/v) sodium azide Recombinant Protein

A 2220 1 ml ANTI-FLAG® M2 Specificity: N-terminal, Met-N-terminal, C-terminal • Immunoprecipitation 5 ml Agarose Affinity Gel FLAG fusion proteins, 3xFLAG fusion proteins • Purification of FLAG 10 ml (Freezer Safe) Binding Capacity: ≥ 0.6 mg per ml gel and 3xFLAG Detection and Purification 25 ml Elution: FLAG peptide; glycine, pH 3.5; 3xFLAG Peptide fusion proteins Form: Suspension of beaded agarose in 50% glycerol containing 10 mM sodium phosphate, 150 mM NaCl, pH 7.4, 0.02% (w/v) sodium azide.

F 2426 1 ml EZview™ Red Specificity: N-terminal, Met-N-terminal, C-terminal FLAG • Immunoprecipitation 5 x 1 ml ANTI-FLAG® fusion proteins, 3xFLAG fusion proteins M2 Affinity Gel Binding Capacity: ≥ 0.6 mg per ml gel Elution: FLAG peptide glycine, pH 3.5; 3xFLAG peptide Form: Suspension of red colored beaded agarose in phosphate buffered saline containing 50% glycerol and 0.0015% Kathon® CG/IPCII as an antimicrobial preservative

FLAG Peptides F 3290 4 mg FLAG® Peptide Sequence: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys • Elution of FLAG fusion 25 mg MW: 1013 proteins from the ANTI- Form: Lyophilized powder FLAG M1 and M2 affinity resins Working Concentration: 100 µg/ml is commonly used to elute FLAG fusion proteins

F 4799 4 mg 3xFLAG™ Peptide Sequence: Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr- • Elution of 3xFLAG fusion 25 mg Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys proteins from ANTI-FLAG Note: The Asp-Tyr-Lys-Xaa-Xaa-Asp motif is repeated M2 affinity gels three times in the peptide; the eight amino acids at the Working Concentration: C-terminus make up the classic FLAG sequence (Asp-Tyr- 100 µg/ml is commonly Lys-Asp-Asp-Asp-Asp-Lys). used to elute 3xFLAG MW: 2861.9 fusion proteins Form: Lyophilized powder sigma-aldrich.com Recombinant Protein 66 Detection and Purification sigma-aldrich.com DETECTION ANDPURIFICATION FLAG AntibodyConjugates Applications Characteristics Description FLAG Antibodies Size Number Package Product F 4042 F 3165 A 8592 A 9469 F 2922 F 9291 F 7425 F 3040 gFLAGfusionproteins expressed inmurinehost,where 5 x1mg gPopaaeFLAGfusionproteins expressed inmurinehost,where Phosphatase 5 x1mg gCnuaeisnotCa Conjugate 5 x1mg 0 gANTI-FLAG 200 µg ANTI-FLAG 200 µg RabbitANTI-FLAG 200 µg ANTI-FLAG 200 µg ANTI-FLAG 200 µg ANTI-FLAG 200 µg 0 gANTI-FLAG 200 µg ANTI-FLAG 200 µg gM-eoiaeof FLAG fusionproteins. Especially usefulindetectionof M2-Peroxidase 1 mg mammaliancrudecellextracts, butnotrecommended cytoplasmicallyexpressed MonoclonalAntibody 5 mg mg 1 gPrfe g ofcalciumions.Doesnotbindto PurifiedIgG 5 mg gPrfe g Ca PurifiedIgG 5 mg 1 mg Monoclonal Antibody, Carboxy-terminal, or internal. Bindingisnot Carboxy-terminal,orinternal. MonoclonalAntibody, 1 mg Ca MonoclonalAntibody, 1 mg gM-laie ofFLAGfusionproteins. Especiallyusefulindetectionof M2-Alkaline 1 mg Monoclonal Antibody, mg 1 C-terminalofFLAGfusionproteins. Binding Antibody, Biotin 1 mg oylnlAtbd Met-N-terminal,andC-terminalFLAG Polyclonal Antibody itnCnuaeis Biotin Conjugate ® ® ® ® ® ® ® BioM5 BioM2 M5 M2 M1 ® al H74 otiig00%sdu zd. Westernblotting NaCl, pH7.4,containing0.02%sodiumazide. Form: expressed proteins. reactivity. BindingisnotCa secondary anti-mouseantibodiesmay causecross- for fusionproteins expressed in not Ca aie H74 otiig1 oie immunofluorescence serum albuminand15mMsodiumazide. saline, pH7.4,containing1%bovine antibody in10mMphosphatebuffered containing 0.02%sodiumazide phosphate, 150mMNaCl,pH7.4, Form: proteins; willnotrecognize unprocessed, Form: reactivity. BindingisnotCa secondary anti-mouseantibodiesmaycausecross- Binding isnotCa Specificity: Specificity: Specificity: Specificity: Specificity: ojgt rti ocnrto sapoiaey1m/l i conjugate protein concentrationisapproximately 1mg/ml. Specificity: approximately 1mg/ml. (chemiluminescent) Specificity: NaCl, pH7.4,containing0.02%sodiumazide. approximately 1mg/ml. Ca fusion proteins. Bindingisnot Specificity: NaCl, pH7.4,containing0.02%sodiumazide. oimaie h ojgt rti ocnrto s (chemiluminescent) Westernblotting sodium azide.Theconjugateprotein concentrationis phosphate, 150mMNaCl,pH7.4,containing0.02% Form: Form: proteins in 50% glycerol pluspreservative andstabilizer. The Form: Form: preservative. Theconjugateprotein concentration is saline containing50%glycerol plusstabilizerand Form: not 2+ 2+ 2+ -dependent. - dependent;thecomplexdissociatesinabsence -dependent. 2+ recommended fordetectionofFLAGfusion Solution in10mMsodiumphosphate,150 Solution in10mMsodium Solution ofaffinity isolated Solution in50%glycerol, 10mMsodium ouini 0m oimpopae 5 M Western blotting Solution in10mMsodiumphosphate,150 Solution in10mMsodiumphosphate,150 Solution inphosphatebuffered salinecontaining Purified immunoglobulinsolutioninTris buffered -dependent. 2+ -dependent. E. coli. Met-N-terminal FLAG.Usefulfordetecting Met-N-terminal N-terminal, Met-N-terminalorC-terminal Met-N-terminal FLAGfusionproteins. N-terminal, Met-N-terminalor N-terminal, Met-N-terminal, N-terminal FLAG.Bindingis N-terminal, Met-N-terminal,orC-terminal Reacts withN-terminal, 2+ -dependent. ANTI-FLAGBioM5 Met-FLAG fusionproteins in 2+ 2+ -dependent. -dependent. E. coli. Met-FLAG fusion idn sW Binding is cytoplasmically • Working Dilution: • • • • • Working Dilution: • • • • • • Working Dilution: • • • Working Dilution: • • Working Dilution: • • • • • • • • • • • • Working Dilution: • • Working Dilution: • • Working Dilution: Western blotting Western Western blotting Western Western blotting Western 10µg/mlbyindirect blotting Western EIA ELISA Immunohistochemistry blotting Western blotting Western 10µg/mlbyindirect Immunocytochemistry blotting Western Western blotting Western EIA 2.5µg/mlbyindirect 5µg/mlbyindirect 10µg/mlbyindirect Immunocytochemistry Dotblotting 1:20,000byindirect ELISA Immunocytochemistry Immunoprecipitation Immunoprecipitation ELISA Immunocytochemistry Immunoprecipitation Immunocytochemistry 10µg/mlbyindirect Immunocytochemistry 2µg/mlbyindirect 1:100-1:1,000by 1:20,000byindirect ELISA Western blotting Western blotting (chemiluminescent) (chemiluminescent) (chemiluminescent) (chemiluminescent) mmunocytochemistry estern blotting estern 67 DETECTION AND PURIFICATION

Product Package Number Size Description Characteristics Applications

FLAG Antibody Conjugates cont’d F 4049 200 µg ANTI-FLAG® M2 Specificity: N-terminal, Met-N-terminal • Fluorescent 1 mg Monoclonal or C-terminal of FLAG fusion proteins. immunocytochemistry 5 x 1 mg Antibody-FITC Binding is not Ca2+-dependent. • Fluorescent Form: Solution in 10 mM sodium phosphate, immunohistochemistry 150 mM NaCl, 1% bovine serum albumin, • Flow cytometry 0.1% sodium azide, pH 7.4. Working Dilution: • 10 µg/ml by indirect immunofluorescence using mammalian cells fixed with methanol:acetone

A 9594 200 µg ANTI-FLAG® Specificity: N-terminal, Met-N-terminal or C-terminal • Immunocytochemistry 1 mg M2-Cy3™ Conjugate of FLAG fusion proteins. Especially useful in detection of Working Dilution: 5 x 1 mg FLAG fusion proteins expressed in murine host, where • 10 µg/ml by direct secondary anti-mouse antibodies may cause cross- immunofluorescence reactivity. Binding is not Ca2+-dependent. using mammalian Form: Purified immunoglobulin in phosphate buffered cells fixed with

saline plus 1% BSA and preservative. The conjugate methanol:acetone Recombinant Protein protein concentration is approximately 1 mg/ml. Detection and Purification ANTI-FLAG Secondary Antibodies A 9044 2 ml Rabbit Anti-Mouse Specificity: Mouse IgG Working Dilution: IgG, (whole molecule) Binds all mouse Igs. • 1:6,000-8,000 by dot blotting Peroxidase Conjugate Form: Solution in 0.01 M phosphate buffered • 1:40,000 by direct ELISA saline, pH 7.4, containing 0.01% • 1:200 by immunohisto- thimerosal as a preservative. chemistry (formalin-fixed, paraffin-embedded sections) • 1:80,000 by indirect Western blotting (chemiluminescent)

A 9917 1 ml Goat Anti-Mouse IgG, Specificity: Mouse IgG Fab Working Dilution: Fab Fragment Immunospecific purification removes essentially all • 1:8,000 by dot blotting Peroxidase Conjugate, goat serum proteins, including immunoglobulins, • 1:60,000 by direct ELISA Adsorbed with which do not specifically bind to the Fab fragment of • 1:150 by immunohisto- Human IgG mouse IgG. The antibody preparation is solid phase chemistry (formalin-fixed, adsorbed with human IgG to ensure minimal cross paraffin-embedded sections) reactivity in tissue or cell preparations. • 1:80,000 by indirect Form: Solution in 0.01 M phosphate buffered saline, Western blotting pH 7.4, containing 0.01% thimerosal as a preservative. (chemiluminescent)

A 3682 1 ml Goat Anti-Mouse IgG, Specificity: Mouse IgG Fab Fragment Immunospecific purification removes essentially all goat Working Dilution: Peroxidase Conjugate, serum proteins, including immunoglobulins, which do not • 1:4,000 by dot blotting Adsorbed with specifically bind to the Fab fragment of mouse IgG. The • 1:40,000 by direct ELISA Human IgG and Rat antibody preparation is solid phase adsorbed with human • 1:150 by immunohisto- Serum Proteins IgG and rat serum proteins to ensure minimal cross chemistry (formalin-fixed, reactivity in tissue or cell preparations. Binds all Mouse Igs. paraffin-embedded sections) Form: Solution in 0.01 M phosphate buffered saline pH • 1:80,000 by indirect 7.4, containing 0.01% thimerosal. Western blotting (chemiluminescent) sigma-aldrich.com Recombinant Protein 68 Detection and Purification sigma-aldrich.com DETECTION ANDPURIFICATION ubrSz ecito hrceitc Applications Characteristics Description FLAG-Control Proteins Size Number Package Product P 7457 P 2104 P 7582 P 5975 . gAiotria A467aminoacidN-terminalFLAG Amino-terminal 0.1 mg A466aminoacidC-terminalFLAG Carboxy-terminal 0.1 mg A482aminoacidN-terminalFLAGfusionprotein Amino-terminal 0.1 mg Met-FLAG 0.1 mg oto rti hshts BP. monoclonalantibodies phosphatase(BAP). Control Protein FLAG-BAP phosphatase(BAP). Control Protein FLAG-BAP Control Protein Met-3xFLAG-BAP oto rti sa48aioai -emnlMtFA FLAGM2andM5 is a 468 amino acid N-terminalMet-FLAG Control Protein ™ ® ™ A N-terminalMet-FLAG-BAPcontrol protein BAP fusionprotein of ™ MW: MW: fusion protein of MW: of hshts BP Westernblotting,ELISA, MW phosphatase (BAP) fusion protein of E. coli 94ka immunoprecipitation, : 49.4kDa. 49.3 kDa. in Western blotting, inWestern procedures suchas 49.3 kDa. 49.1 kDa. 49.9 kDa. atra laiepopaae(A) for bacterial alkalinephosphatase(BAP). E. coli E. coli E. coli atra laie ANTI-FLAGM1andM2 bacterial alkaline bacterial alkaline bacterial alkaline • • • • Control protein for Control protein forthe Control protein Control protein forANTI- and inimmunoaffinity light microscopy, FACS, fluorescence microscopy, ELISA, immunoprecipitation, and immunoaffinity light microscopy, FACS, fluorescence microscopy, immunoprecipitation, Western blotting,ELISA, antibody inimmunological and immunoprecipitation, WesternELISA, blotting, antibodyin monoclonal ANTI-FLAG M2monoclonal chromatography withthe and inimmunoaffinity light microscopy, FACS fluorescence microscopy, monoclonal antibodiesin chromatography ANTI-FLAG M2affinity gel chromatography chromatography ANTI-FLAG M2 immunoaffinity 69 DETECTION AND PURIFICATION

FLAG® Purification FLAG-Fusion Protein Detection Using StarBright® Green Substrate ANTI-FLAG® HS M2 Coated Plates 6 x 105

ANTI-FLAG High Sensitivity 96-well plates provides a convenient, ready to use, 5 x 105 platform for the capture and detection of FLAG® fusion proteins. The ANTI-FLAG 4 x 105 M2 coating can detect as little as 1 ng/well (Figure 1) with a capacity of up to 300 5 ng/well of FLAG fusion protein. Suitable for screening for expression, study of RFU 3 x 10 protein:protein interactions, and ELISA assays. The ANTI-FLAG high sensitivity M2 2 x 105 coated multiwell plate can be used to detect N-terminal, Met-N-terminal, internal, 1 x 105 and C-terminal FLAG and 3xFLAG fusion proteins. 0 0.1 1 10 100 1000 Nanogram of FLAG-BAP/well

Figure 1: Ten microliters of N-terminal FLAG-BAP Control Product Code Description Size protein (Prod. Code P 7582) diluted in Tris buffer with 1% BSA was incubated in an Anti-FLAG M2 coated clear 96-well P 2983 ANTI-FLAG High Sensitivity Clear M2 1 each plate (Prod. Code P 2983) for two hours at room tempera- coated 96-well plate 5 each ture. The plate was washed four times with TBS with 0.05% TWEEN®-20 on a plate washer. One hundred microliters of StarBright® Green substrate was added and the reaction was incubated at 37 ºC for 10 minutes. Fluorescence was read on a Wallace Victor2™ plate reader. Results demonstrate that the ANTI-FLAG HS M2 coated plates can purify as little as 1 ng of fusion protein.

FLAG® M Purification Kit Components Recombinant Protein The FLAG M Purification Kit utilizes CelLytic™ M for rapid and efficient lysis, and CelLytic M ® protein extraction from mammalian cells, and the ANTI-FLAG M2 affinity gel for 10x Wash Buffer Detection and Purification affinity purification of active FLAG-tagged proteins. It can also be used for immuno- Elution Buffer precipitation. The affinity purification is performed with ANTI-FLAG M2 affinity gel, 3xFLAG Peptide which is a highly specific monoclonal antibody covalently attached to agarose resin. The use of an affinity resin allows for efficient binding of FLAG-tagged proteins ANTI-FLAG M2-Agarose Affinity Gel without the need for preliminary steps and calibrations. The affinity bound FLAG- Amino-terminal FLAG-BAP Fusion Protein tagged proteins can be efficiently eluted from the resin by acidic conditions or by Polypropylene chromatography column competition with 3xFLAG® peptide. The eluted proteins can be analyzed for their activity, size, post-translational modifications, interactions, etc. Sufficient for 3-5 uses of 1 ml affinity purification column.

Product Code Description Size CelL-M-M2 FLAG M Purification Kit 1 kit

® FLAG Y Purification Kit Components The FLAG Y Purification Kit utilizes CelLytic™ Y for rapid and efficient lysis, and CelLytic Y ® protein extraction from yeast cells, and the ANTI-FLAG M2 affinity gel for affinity 10x Wash Buffer purification of active FLAG-tagged proteins. The affinity purification and immuno- Elution Buffer precipitation is performed with ANTI-FLAG M2 affinity gel, which is a highly specific 3xFLAG Peptide monoclonal antibody covalently attached to agarose resin. The use of an affinity resin allows for efficient binding of FLAG-tagged proteins without the need for ANTI-FLAG M2-Agarose Affinity Gel preliminary steps and calibrations. The affinity bound FLAG-tagged proteins can be Amino-terminal FLAG-BAP Fusion Protein efficiently eluted from the resin by acidic conditions or by competition with 3xFLAG® Polypropylene chromatography column peptide. The eluted proteins can be analyzed for their activity, size, post-translational modifications, interactions, etc. Sufficient for 50 immunoprecipitation reactions or one 1 ml affinity purification column.

Product Code Description Size CelL-Y-M2 FLAG Y Purification Kit 1 kit sigma-aldrich.com Recombinant Protein 70 Detection and Purification sigma-aldrich.com DETECTION ANDPURIFICATION Tris Buffered SalinewithTWEEN Tris Buffered Saline,pH8.0with3%nonfatmilk Reaction Buffer Chemiluminescent Peroxidase Substrate(CPS) (CPS) Reagent Chemiluminescent Peroxidase Substrate ANTI-FLAG-M2-Peroxidase (HRP)Conjugate N-FLAG-BAP Control protein Components 3,3 ANTI-FLAG-M2-Peroxidase (HRP)Conjugate N-FLAG-BAP Control protein Components ’ 5,5 ’ -Tetramethylbezidine (TMB)Substrate ® -20 25 mini-gelsized(10x10cm)blots. immunoprecipitation samplesontheblot.Thiskithassufficient reagents for the ANTI-FLAG-HRPconjugateandheavylightantibodychainsin blots from immunoprecipitation experiments,becausethere isnoreaction between idase conjugates.Inaddition,superiorresults canbeobtainedforimmunostaining unconjugated anti-FLAGantibodieswithanti-mouseIgGsecondaryantibodyperox- non-specific background andsimplifiestheprocedure compared totheuseof sufficient reagents forimmunostaining12mini-gelsized(10x10cm)blots. ies withanti-mouseIgGsecondaryantibodyperoxidase conjugates.Thekithas simplifies theprocedure compared totheuseofunconjugatedANTI-FLAGantibod- monoclonal antibody-peroxidase conjugateeliminatesnon-specificbackground and ProteoQwest film orimagerisneeded.ApurifiedFLAG-taggedfusionprotein, N-FLAG-BAP membrane andcanbevisuallymonitored fordesired signalintensity;nodarkroom, fusion protein blots.Thecolorimetricreaction onWestern occursdirectly onthe Designed forcolorimetricdetectionofaslittle1ngFLAGepitope-tagged ProteoQwest procedure hasbeenoptimized.Immunostainingwiththeprovided ANTI-FLAG FLAG-tagged protein. Allofthecomponentskithavebeentestedand kit provides highsensitivitychemiluminescentdetectionofaslittle0.1ng blots.Thechemiluminescentperoxidaseon Western substrateincludedinthe Designed forchemiluminescentdetectionofFLAGepitope-taggedfusionprotein the provided ANTI-FLAG has beenincludedtoconfirmproper performanceofthekit.Immunostainingwith Western BlottingKit,CPSSubstrate Western Blotting Kit,TMBSubstrate rdc oeDsrpinSize PQ0300 Description Product Code rdc oeDsrpinSize PQ0400 Description Product Code rtows LGClrmti etr 1 kit Blotting Kit,TMBSubstrate ProteoQwest FLAGColorimetricWestern rtows LGCeiuiecn 1kit Western BlottingKit,CPSSubstrate ProteoQwest FLAGChemiluminescent ™ ™ ® M2 monoclonalantibody-peroxidase conjugateeliminates FLAG FLAG ® ® Chemiluminescent Colorimetric Western ® ™ M2 , 71 DETECTION AND PURIFICATION

® FLAG Immunoprecipitation 1. Lyse cells, add affinity resin FLAG® Immunoprecipitation Kit Sigma’s FLAG Immunoprecipitation Kit provides a rapid and efficient immunoprecipitation and elution of an active FLAG-tagged protein (Fig. 1). Typically 70-90% of a bound FLAG fusion protein is released and retains biological activity. Immunoprecipitation is a powerful technique for the isolation of proteins or protein complexes. Immunoprecipitation consists of the following steps: cell lysis, 2. Incubate to allow complex formation binding of specific antigen to an antibody, antibody-antigen complex precipitation, precipitant wash and antigen dissociation from the immune complex. Epitope- tagged proteins can be affinity purified and immunoprecipitated, using highly spe- cific antibodies raised against their epitope. The use of such antibodies facilitates subsequent biochemical and immunological analysis. 3. Spin Features & Benefits • Utilizes highly specific ANTI-FLAG® M2 affinity gel • No preliminary steps or calibrations • 3xFLAG® peptide provides easy and gentle elution through direct competition

Components

4. Wash Recombinant Protein ANTI-FLAG M2 affinity gel Elution Buffer FLAG-BAP™ Control Protein Detection and Purification 3xFLAG Peptide Lysis Buffer 2x Sample Buffer 5. Elute 10x Wash Buffer

Product Code Description Size FLAG-IPT-1 FLAG Immunoprecipitation Kit 1 kit PAGE Western MALDI Blot Mass Spec.

Figure 1. Affinity-based molecular pull-down technique. sigma-aldrich.com Recombinant Protein 72 Detection and Purification sigma-aldrich.com detection ofprotein-protein interactionsinahighthroughput format. step ofexpression. TheIPDetectionkitsincludetheingredients forcolorimetric fusion.Control plasmidsandverificationprimersare includedtoverifyeach c-Myc expression vectors;oneforaN-terminalFLAGfusion,theother appropriate forgreatest flexibility. Thecore kit( within thesystemare optimizedforusetogether, yetcanbeusedindividuallyas designed specificallyforCo-IPandvalidationofprotein-protein interactions.Kits • • • • Features &Benefits The core kit( antibody. colorimetric assayusingananti-c-Myc M2 HighSensitivity96-Well Plate.Finally, theinteractingprotein isdetectedbya epitopes.Thebaitprotein isthencaptured ontheANTI-FLAG the FLAGandc-Myc required whenperformingIPassays. Potentialbindingpartnersare firsttaggedwith dling ofmultipleIPsamplesandeliminatethecentrifugationstepstypically The FLAG96-Well Immunoprecipitation (IP)Systemisdesignedtofacilitatethehan- Accurately QuantitateProtein-ProteinInteractionsina96-Well Format FLAG DETECTION ANDPURIFICATION performed inanELISAprocedureusingalkalinephosphatase-conjugatedanti-c-Mycantibody (1:100). amounts ofcelllysatewereincubatedwiththewellsFLAGM2plate.Detection exogenouslargeTantigenwas Figure 2. for amultitudeofuserdefinedapplications.TheIPVector kit( preventing anyinterference blots onWestern ANTI-FLAG M2antibodyiscovalentlycoupledtothesurfaceofwell, ELISAformatforprotein-proteinTimely interactions 96-well formatgreatly increases throughput 3 Kitsmakeupanintegratedandcompletesystemyetallowflexibility ELISA AnalysisoftheintereactionFLAG-p53andc-Myc-largeTAntigencaptured on ANTI-FLAGM2Platesshowingquantitativedetectionofc-MyclargeTantigen. 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 1 COS-7 cellswereco-transfectedwithFLAG-p53andc-Myc-largeTantigen and c-Myc-GFP. Various 5 01 2 5 3035 2025 10 15 5 0 ® 96-Well Immunoprecipitation System HT -COIP-1 FLAG-p53 +c-Myc-largeTantigen buffer alone ), IPVector ( COIP-P ) andIPDetection( HT -COIP-1 FLAG-p53 +c-Myc-GFP ) alonecanbethebasis COIP-D COIP-P ) kitsare ) includestwo ® kDa c-Myc tagonthelargeTantigen. captured viatheFLAGtagonp53anddetected antibody. Theinteractingp53-largeTantigencomplexwas were subjectedtoWestern blottingwithanti- incubated inthewellsofFLAGM2plate.Elutedproteins fected withFLAG-p53andc-Myc-largeTantigen,were Cell lysates,preparedfromCOS-7cellsthatwereco-trans- Lane 3:30µgco-IPofcelllysateco-transfectedwith Lane 2:30µgco-IPofcelllysateco-transfectedwith Lane 1:30µgcelllysatewithnoplasmids showing specificdetectionoftheinteraction. c-Myc Co-Immunoprecipitation ofFLAG-p53and Mw 123 203 22 30 36 50 83 7 Western blotusingAnti-c-Myc -large TantigenonFLAGM2plate FLAG-p53 andc-Myc-largeTantigen FLAG-p53 andc-Myc-GFP 12 3 c-Myc T antigen c-Myc-large 73 DETECTION AND PURIFICATION

Components Immunoprecipitation Vector Kit pFLAG-CMV-2 Expression Vector The Immunoprecipitation Vector Kit is for use with the FLAG® 96-Well pc-Myc-CMV-2 Expression Vector Immunoprecipitation System. The kit provides expression vectors pFLAG-CMV™-2 pFLAG-CMV-2-p53 control plasmid and pc-Myc-CMV™-2 designed for the cloning and expression of protein interac- tion candidates as N-terminal FLAG® and N-terminal c-Myc fusions. Control plasmids pc-Myc-CMV-2-Large T Antigen pFLAG-CMV™-2-p53, pC-Myc-CMV-2-Large T antigen and pC-Myc-CMV-2-BAP control plasmid are supplied with the expression vectors. The control plasmids are intended for pc-Myc-CMV-2 BAP control plasmid expression of positive and negative binding partners in the immunoprecipitation Verification Primer–MF analysis. The kit also includes the MF-2 and MR-2 verification primers. Verification Primer–MR 4679 Hind III 4688 Hind III Not I 188 Not I 166 4227 EcoR I 4236 EcoR I f1 IG Cla I f1 IG Cla I Bgl II CMV Bgl II CMV EcoR V EcoR V Kpn I Kpn I FLAG c-MYC Sal I Sal I Xba I Xba I pFLAG-CMV-2 Bam HI pc-Myc-CMV-2 Bam HI 4.7 kb Sma I 4.7 kb Sma I hGH poly A hGH poly A

1642 1651

SV40 ori SV40 ori E.coli orilb-lact E.coli orilb-lact 2908 2917

Product Code Description Size COIP-P Immunoprecipitation Vector Kit 1 kit Recombinant Protein Detection and Purification

Components FLAG® 96-Well Immunoprecipitation Kit ANTI-FLAG M2 Capture Plate The FLAG 96-Well Immunoprecipitation Kit is the core kit of a system to validate Amino-terminal FLAG-BAP fusion protein protein-protein interactions in a convenient 96-well format. The kit contains the ® 10x Wash Buffer ANTI-FLAG 96-well plate and all reagents necessary for capture and elution of FLAG fusion proteins. This flexible kit can be the basis for user defined assays as Lysis Buffer well as use as part of the FLAG 96-well IP System. 2x Sample Buffer SealPlate Film Product Code Description Size HT-COIP-1 FLAG 96-Well Immunoprecipitation Kit 1 kit

Components Immunoprecipitation Detection Kit Monoclonal Anti-c-Myc, Clone 9E10, The Immunoprecipitation Detection Kit contains the reagents needed for colorimetric Alkaline Phosphatase Conjugate detection of protein-protein interactions on the 96-well ANTI-FLAG® M2 plate. The Tris-Buffered saline, pH 8.0, with supplied Anti-c-Myc alkaline phosphatase conjugated antibody recognizes and binds 3% Nonfat Milk to the c-Myc epitope tag on the co-expressed binding partner of the FLAG-tagged SIGMA FAST™ p-nitrophenyl fusion protein immobilized on the plate. The conjugated antibody hydrolyzes the Phosphate Tablets supplied p-Nitrophenyl Phosphate Disodium (pNPP) substrate to produce a yellow- colored end product that can be read spectrophotometrically at 405 nm.

Product Code Description Size COIP-D Immunoprecipitation Detection Kit 1 kit sigma-aldrich.com