intensity followingelectrophoresis andCoomassie formulated toyieldadistributionofwell-definedbandsapproximately equal low rangemarkersare ready forusefollowingreconstitution withwater. Theyare proteins andtheirsubunits.Lyophilized withsamplebuffer, thewide,high,and SigmaMarkers encompasstherangeofmolecularweightscommontomost SigmaMarker™ Recombinant proteins formsharpbandsforaccuratesampleMWcalculations. precisely sizedproteins, molecularweights15,25,35,50,75,100,and150kDa. The RecombinantMolecularWeight Standard Mixture containsseven Recombinant Markers ELECTROPHORESIS rdc oe ecito MWRange(Da) M M M Description Product Code MWRange(Da) M 0671 Description Product Code 3788 3913 4038 SigmaMarker Wide (6,500-205,000) Wide SigmaMarker High SigmaMarker Low SigmaMarker (36,000-205,000) (6,500-66,000) 15,000-150,000 Standard Mixture Recombinant MolecularWeight ® Blue staining. M 4038 4-20% PAGE gelandstainedwithEZBlue ( , M 3913 , and M 3788 with EZBlue™( 4-20% PAGE gelandstained M 0671 (5 µl)wererunona (5 µl)wasrunona G 1041 G 1041 ). ).

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Protein Analysis 103 Electrophoresis Protein Analysis 104 Electrophoresis sigma-aldrich.com ELECTROPHORESIS silver-stained. wererunona10-18%SDS-PAGE geland dilution) M 5630 , M 6539 , and M 5505 M 3546 ( stained withEZBlue™ with 5%glutaraldehydeand Tris-Tricine SDS-PAGE gel,fixed tion) wasrunona10-20% G 1041 (7.5 µlofa1:15 ). (5 µlofa1:20dilu- molecular weightproteins. Tris-Tricine SDS-PAGE systems.Glutaraldehydefixingissuggestedtoretain low Sigma’s Ultra-LowRangemolecularweightmarkerisrecommended forusewith Ultra-Low RangeMarkers resolved proteins ofequalintensity. SDS-PAGE molecularweightstandard mixtures containevenlydistributed,well Designed formolecularweightdeterminationsonsilverstainedgels,SilverStain Silver StainMarkers M M rdc oe ecito MWRange(Da) M Description Product Code rdc oe ecito MWRange(Da) M Description Product Code 5505 6539 5630 3546 ivrSanSSPG High29,000-116,000 6,500-180,000 Wide Low14,000-66,000 Molecular Weight Standard Silver StainSDS-PAGE Molecular Weight Standard Silver StainSDS-PAGE Molecular Weight Standard Silver StainSDS-PAGE oeua egtMre o Ultra-Low1,000-26,600 SDS-PAGE Molecular Weight Markerfor See alsoProteoSilver ™ , page110. Protein DyesandStainsSelectionChart EZBlue™ andProteoSilver™, designedspecificallyforproteomics, alsoperformimpressively intraditionalPAGE formats. To meetthegreat diversityofprotein analysisneeds,Sigmaoffers awideselectionofprotein visualization(staining)reagen Protein StainingReagents ELECTROPHORESIS Detection Special Features Compatibility Method Targets Phospholipids, NeutralFats,&Sterols BEST Choice Good Choice Highest Sensitivity(0.1ng/mm Rapid Development(5minutes) Cellulose AcetateMembrane Lipids and/orLipoproteins Nitrocellulose Membrane Higher Sensitivity(1ng) Requires NoDestaining High Sensitivity(10ng) Nylon Membrane PVDF Membrane IEF (Acrylamide) Fixing Reagent Glycoproteins Nucleic Acids Colorimetric Agarose Gel Fluorescent MALDI-MS Reversible Proteins PAGE 2 )

Alcian Blue 8GX — A 3157 Amido Black Staining Solution — A 8181 Brilliant Blue G Concentrate — B 8522 Brilliant Blue R Staining Solution — B 6529 Coomassie™ Violet R 200 — 27817 Eosin Y — 45235 EZBlue™ — G 1041 Fast Green FCF — F 7252 Fixing Solution — F 7264 Fluorescamine — F 9015 GlycoProfile III — PP0300 GlycoProtein Detection Kit (Colorimetric) — GLYCO-PRO Gold Solution, Colloidal — 50755 Oil Red O — O 9755 Ponceau S Solution — P 7170 ProteoSilver™ — PROT-SIL1 ProteoSilver Plus — PROT-SIL2 Reversible Protein Detection Kit — R-PROB Sudan Black — 86015 SYPRO® Orange Protein Gel Stain — S 5692 SYPRO® Red Protein Gel Stain — S 5817 SYPRO® Ruby Protein Blot Stain — S 4817 ® SYPRO Ruby Protein Gel Stain — S 4942 ts. SYPRO® Tangerine Protein Gel Stain — S 5942

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Protein Analysis 105 Electrophoresis Protein Analysis 106 Electrophoresis sigma-aldrich.com Sigma EZBlue( Following electrophoresis,thegelwasstained with diluted andrunonaTris-Acetate 6-15%gel. carbonic anhydrase. demonstrates detectionof5ngBSAand11 ngof SigmaMarker™, Wide Range( SigmaMarker™, Wide ELECTROPHORESIS protein gel Separated G 1041 (total 15minutes)withagitation. Agitate andincubatefor1hour. remove thegelfromitscassette to intensifyproteinbands. Following electrophoresis, water, for5minuteseach Add EZBlue for 1hourtoovernight ). Thelanetothefarright Wash gel3timesin (process complete) and placeintray. Rinse withwater Optional: Decant Decant

stain reagent. M 4038 ) wasserially H H 2 2 O O • • Brilliant BlueGConcentrate • • Features &Benefits sensitive, detectingaslittle5ngofprotein. intensify bandsandclarifythebackground. Mostimpressively, EZBlueisextremely almost immediately. Nodestainingstepisrequired, althoughawaterwashmay the gelitself.Background stainingisreduced, soprotein bandscanbevisualized additions ofmethanoloracid.Asacolloidalstain,itreacts onlywithproteins, not staining time.Convenientlypackaged,EZBluerequires nomessyweigh-upsor protein stainimproves protein electrophoresis results whilesignificantlyreducing Convenient, sensitive,andsafe,EZBlueCoomassieBrilliantBlueG-250colloidal EZBlue™ GelStainingReagent Coomassie™ Stains of carrierampholytes. R200isusedforrapiddetectionofproteinsCoomassie Violet inIEFwithoutremoval Coomassie™ Violet R200 for 30minutesanddestainedwith10%aceticacid. gels require nofixingsteppriortostaining.Thegelisimmersedinstainingsolution SDS-PAGE andagarose gels.Becausethisstaincontainsethanolandaceticacid, Brilliant BlueRStainingSolutionisdesignedspecificallyforstainingprotein in Brilliant BlueRStainingSolution dilutes to1Lwithwater. methanol andaceticacid,gelsrequire nofixingsteppriortostaining.Eachbottle for protein detectioninpolyacrylamideandagarose gels.Becausethisstaincontains Brilliant BlueGConcentrateisaCoomassieG-250methanol-basedstaindesigned 27817 rdc oe ecito Size B 6529 B 8522 G 1041 Description Product Code material disposal Eliminates solventwaste and rinse Rapid reaction (as littleas5ng) Increased sensitivity the stain Pre-mixed solution significantly reduces theamountoftimerequired tostain omsi iltR2025g 1L 1Btl 500 mL R200 Coomassie Violet Brilliant BlueRStainingSolution Brilliant BlueGConcentrate EZBlue GelStainingReagent eliminates thetimeandeffort required toprepare ensures lowabundanceproteins canbedetected so yousavetheexpenseofhazardous 100 g 3.8 L a 4-20%gelandstainedwithProteoSilver( Serial dilutionsofSigmaMarker( ELECTROPHORESIS and 10rangefrom0.3-1ng. 1:1000. Theamountsofproteinforeachbandinlanes9 and 8:5µldiluted1:1000.Lanes910:2.5 diluted 1:100.Lanes5and6:10µl1:1000.7 Lanes 1and2:5µldiluted1:100.34:2.5 Both kitscontainsufficient reagents forstaining25mini-gels. perform furthercharacterizationthrough trypticdigestandMALDI-MSanalysis. two additionalreagents fordestaininganexcisedprotein spot,forthosewishingto which crosslinks lysineresidues, resulting ininaccuratespectra.Thiskitcontains ProteoSilver PlusisMALDI-MScompatibleanddoesnotcontainglutaraldehyde, ground, ProteoSilver leadstosuperiordetectionoflowabundanceproteins. • • • Features &Benefits adetectionlimitof0.1ng/mm With silver stainingreagents alongwithdetailedinstructionstoachieveoptimalresults. ideal products foranyproteomics scientist.Bothkitscontainprepared solutionsof Conveniently packaged,andhighlysensitive,ProteoSilver andProteoSilver Plusare ProteoSilver™ andProteoSilver Plus rdc oe ecito Size PROT PROT Description Product Code mass spectrometry usingProteoSilver Plus MALDI-compatible. be detectedandresolved from otherproteins High sensitivityandlowbackground preparing individualcomponents Pre-mixed andpre-weighed -SIL2 -SIL1 rtoivr1kit 1kit ProteoSilver Plus ProteoSilver M 4038 Protein spotsofinterest canbefurthercharacterizedby ) wereloadedonto PROT -SIL1 solutions reduce timeandcostofpurchasing and ). 2 of protein (BSA)andanextremely lowback- ensure verylowabundanceproteins can using ProteoGelIPGEquilibrationBuffer( a ProteoGel™IPGstrip(pH3-10)( 4 µgofE.coliwholecellextractwasseparatedbyIEFusing the gelwasstainedwithProteoSilver( on aTris-Acetate 2D8-18%gel.Followingelectrophoresis, I 2531 PROT ), equilibrated I 7281 -SIL1 ), andrun ). ProteoSilver Plus ProteoSilver

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Protein Analysis 107 Electrophoresis Protein Analysis 108 Electrophoresis sigma-aldrich.com ELECTROPHORESIS were analyzedinthelinearpositiveionmodeonaKratos and 1µlwasspottedontheMALDItarget.Thepeptides destained usingProteoSilverPlus( Kompact SIL2 SDS-PAGE gelandsilverstainedwithProteoSilverPlus( Bovine carbonicanhydrase(220ng)wasrunona4-20% equal volumeof The peptideswereconcentrated(3µl),combinedwithan Grade Trypsin ( the excisedbandwasdigestedovernightwithProteomics were generated.Nointerferencewasobserved. ). Thesilverstainedbandwasexcisedfromthegeland ProteoSilver Plus ® MALDI-MS. Onlytheexpectedpeptidefragments [Proteomics SequencingGradeTrypsin( T 6567 Destaining a -cyano-4-hydroxycinnamic acid( Ready fortrypticdigestand 2X ), andthepeptideswereextracted. MALDI-MS analysis Incubate atRT supernatant Centrifuge Excise spot or band Decant Decant Vortex 2 min 5 sec PROT Destainer B Destainer A H 2 -SIL2 O ). Theproteinin T 6567 )] C 8982 PROT ® ) - ProteoSilver™ andProteoSilver PlusStaining ( imaged, andthenstainedfortotalproteinwithEZBlue™Gelstainingreagent SDS-PAGE gel.Thegelwasstained withGlycoProfileIII(PP0300)(left),fluorescently and non-glycosylatedproteins,wasseparatedbyelectrophoresisona4-20% gels. Ex/Em=280,450/610nm especicially for2DPAGE, SYPRORubyalso performsimpressively inPAGE andIEF luminescent stainforthedetectionofproteins separatedbyPAGE. Designed • • • • • Features &Benefits Molecular Probes 8 Superior Selectivity ELECTROPHORESIS ProteoProfile PTM Marker(Product Code glycoprotein detectionproduct available. this research. Sigma understandsandmeetsyourneedswiththemostselective research. Accurateinitialidentification ofglycoproteins playsanimportantrole in Glycoprotein study, orGlycomics,isarapidlygrowing, dynamicnewfieldof PP0300 phosphorylated andunmodifiedproteins andisavailableindividuallyaswellwith Identify glycoproteins withsuperiorselectivityusingSigma Fluorescent Glycoprotein DetectionKit GlycoProfile™ III 8 *Refer toPost-Translational Modificationsection,p20-24. G 1041 rdc oe ecito Size S 4942 Description Product Code rdc oe ecito Size P 1745 PP0300 Description Product Code Sensitive to1ngofprotein notstainextraneousnucleicacids Will and others Stains glycoproteins, lipoproteins, calciumbinding proteins, fibrillarproteins Delivers alinearquantitationrange overthree orders ofmagnitude Uses asimplestainingprotocol withnopossibilityofover-staining lcPoieII EZBlue GlycoProfile III ) (right).Eachbandrepresentsapproximately300ngofprotein. ( PP0300 . SYPRO )( ProteoProfile PTMMarker( ’ SYPRORubygelstainisaready-to-use, ultrasensitive, YR uyGlSan1kit SYPRO RubyGel Stain ProteoProfile GlycoProfile G 1041 ® Ruby GelStain ™ ) b Ovalbumin (glycosylatedandphosphorylated) BSA (notglycosylated,notphosphorylated) RNase B(glycosylated,notphosphorylated)

-Casein (notglycosylated,phosphorylated) ™ ™ P 1745 IIFursetGyorti eeto i 1kit Kit IIIFluorescent Glycoprotein Detection PMMre 0.1ml PTMMarker ), containingglycosylated P 1745 ) containsamixofglycosylated, ’ s newGlycoProfile III. covered 60-90 minutes, Stain proteins 5 minutes Wash gel 20 minutes carbohydrates Oxidize 2 x30minutes Wash gel minutes the gel60 Fix proteinsin EZBlue proteins with Stain all Em 312nm glycoproteins at View orimage 2 x30minutes Wash gel for easiercomparison withthesilver-stained gel. the gelstainedwithSYPRO Rubydyehavebeeninverted stain (left)orsilver (right).Thegrayscalevaluesof cal 2-Dgelsandstained withSYPRORubyproteingel Proteins fromafibroblast celllysatewererunonidenti- SYPRO Rubyproteingelstaincomparedtoa silver stain. sample(s) toPAGEgel. Apply ProteoProfile Separate proteins. PTM Markerand Optional Decant Decant Decant Decant Decant Decant Stain GlycoProfile Water Ultrapure agent Oxidizing water Ultrapure 50% methanol 3% aceticacid, Water Ultrapure ™ III

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Protein Analysis 109 Electrophoresis Protein Analysis 110 Electrophoresis sigma-aldrich.com the activityofbeta-D-glucuronidase(bottom). bation withELF97D-glucuronidasesubstratetodetect SYPRO Tangerine proteingelstain(top)followedbyincu- gelsandstainedwith on identicalSDS-polyacrylamide amounts ofEscherichiacolibeta-glucuronidasewererun Molecular weightstandardscontainingdecreasing protein patternsusingSYPROTangerine proteingelstain. beta-D-glucuronidase activityaftervisualizationoftotal Sypro Tangerine GelstainZymographicdetectionof ELECTROPHORESIS stains. SYPRO Orange(left)orRed(right)proteingel cal 12%SDS-PAGE gelsandthenstainedwitheither Protein MolecularWeight Standardsseparatedonidenti- • • • Features &Benefits staining ofproteins inelectrophoretic gels. Molecular Probes 8 • • • Features &Benefits blotting. Ex/Em=300,490/640nm proteins tomembranes,allowingvisualizationbefore preceeding withWestern with massspectrometry. Inaddition,stainingdoesnotiterfere withthetransferof for furtheranalysis.Thestaindoesnotalterprotein structure anddoesnotinterfere gel enzymeactivity)assays.Stainedproteins canalsobeelutedfrom gelsandused teins inSDSgels.Proteins stainedwithoutfixationcanbeusedforzymography(in- Molecular Probes 8 nucleic acids. as small6.5kDacanbedetectedwithnoobservedstaining ofliposaccharidesor SYPRO Red,andTangerine) aswellaSYPROphotographicfilter. Proteins The SYPROgelstarterkitincludesthree fluorescent protein stains(SYPROOrange, 8 • • • Features &Benefits staining ofproteins inelectrophoretic gels. Molecular Probes 8 rdc oe ecito Size S 5942 Description Product Code rdc oe ecito Size Size SY0100 Description Product Code Size S 5817 Description Product Code S 5692 Description Product Code tude, muchbroader thanCoomassieorSilverStainingcanprovide Fluorescence intensityislinearwithprotein quantityoverthree orders ofmagni- eliminate negativestaining Minimize protein-to-protein variabilitythrough increased stainingconsistencyand Detect 4-8ngofprotein permini-gelband contaminants Selectively detectsproteins withoutstainingnucleicacidsorlipopolysacharide Staining iscompleteinlessthananhour Detects 4-8ngofprotein permini-gelband tude, muchbroader thanCoomassieorSilverStainingcanprovide Fluorescence intensityislinearwithprotein quantityoverthree orders ofmagni- eliminate negativestaining Minimize protein-to-protein variabilitythrough increased stainingconsistencyand Detect 4-8ngofprotein permini-gelband SYPRO OrangeGelStain SYPRO Tangerine GelStain SYPRO Protein Gel StarterKit SYPRO RedGelStain ’ ’ ’ SYPROOrangeprotein gelstainprovides fast,simple,sensitive SYPRORedprotein gelstainprovides fast,simple,sensitive SYPROTangerine gelstainisanextremely versatilestainforpro- YR agrn e ti 1kit SYPRO Tangerine GelStain YR rti e tre i 1 kit 1kit SYPRO Protein GelStarterKit SYPRO RedGelStain 1kit SYPRO OrangeGelStain drate perband.Eachkitcontains sufficient materials for10mini-gels(10xcm). colorless background. Thedetectionlimitsusing thiskitare 25-100ngofcarbohy- methods. Stainingofsugarmoietiesglycoproteins yieldsmagentabands with a acrylamide gelsandmembranesusingamodificationofthePeriodic Acid-Schiff (PAS) The glycoprotein detectionkitisdesignedtoselectivelystainglycoproteins inpoly- Glycoprotein Detection Kit to formhighly-fluorescent compounds. This reagent reacts readily withprimaryaminogroups inaminoacidsandpeptides Fluorescamine Blue R.ItisespeciallywellsuitedtoIEFbecauseitdoesnotbindampholytes. 625 nm.FastGreen stainingislinearoverawiderrangeofdetectionthanBrilliant PAGE, andIEFgels.Followingstaining,protein concentrationcanbemeasured at Fast Green FCFisusedforprotein detectionandquantitationinnativePAGE, SDS- Fast Green FCF PAGE. Proteins canthenberecovered from thegelandfurthercharacterized. Eosin Yellowish reversibly stainspeptidesandproteins darkred followingSDS- Eosin Y with PAGE gels.Eachbottleof2Xconcentratedilutesto500mlwithwater. isopropanol and10%(v/v)aceticacidforfurtheranalysis.Amidoblackisalsocompatible tration proteins withalowbackground. Proteins canbeeasilydestainedwith25%(v/v) lulose membranes.AmidoBlackStainingSolutionfacilitatesvisualizationoflowconcen- Amido BlackStainingSolutionisdesignedforrapidstainingofprotein bandsonnitrocel- Amido BlackStainingSolution,2Xconcentrate alone react atpH1. by pH;bothsulfateandcarboxylategroups willreact atpH2.5,butsulfategroups The interactionofAlcianBlue(cationic)andpolyanionicglycoproteins isinfluenced PAGE gels.AlcianBlue8GXreacts withsulfateandcarboxylatedfunctionalgroups. Alcian Blue8GXissuitablefordetectionofglycoproteins onnitrocellulose andin Alcian Blue8GX ELECTROPHORESIS rdc oe ecito Size Size GL Description Product Code Size F 9015 Description Product Code Size F 7252 Description Product Code Size 45235 Description Product Code Size A 8181 Description Product Code A 3157 Description Product Code YCO-PRO lcpoenDtcinKt1kit 25mg Glycoprotein DetectionKit 5g Fluorescamine 25g Fast Green FCF Eosin Y 1Btl 10g Amido BlackStainingSolution2xConcentrate Alcian Blue8GX ® 100 mg 100 g 25 g 25 g 1 g 809 pp.,combbound Humana Press,2002, 368 pp.,hardcover Electrophoresis in Wiley-VCH, 2001, Protein Protocols R. Westermeier, Walker, J.M., 2nd Edition 3rd Edition Handbook, Practice, P 4742 E 3402

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Protein Analysis 111 Electrophoresis Protein Analysis 112 Electrophoresis sigma-aldrich.com ELECTROPHORESIS P 2481 Z36,949-7 608 pp.,hardcover Humana Press,1998, Link, A.J., Analysis Protocols 2-D Proteome comb bound 1996, 396pp., Laboratory Press, Cold SpringHarbor D.R. Marshal,etal., Course Manual A Laboratory Characterization: Purification and Strategies forProtein sulfosalicylic acid. (w/v) PonceauSin5%aceticacidor2%30%TCAand well asoncelluloseacetatemembranes.Commonstainformulationsinclude0.1% tion ofprotein bandsonnitrocellulose blotting),as orPVDFmembranes(Western Ponceau S(sodiumsalt)maybeusedtoprepare astainforrapidreversible detec- Ponceau S then destainedinamethanolglycerol solution. ately before staining.Themembraneisstainedforapproximately onehourand stock solutioninmethanolisprepared anddilutedwithsodiumhydroxide immedi- Oil RedOstainissuitableasalipid/lipoprotein stainoncelluloseacetate.A2% Oil RedO membranes followingPAGE. colloidal goldisausefulgeneralstainforproteins immobilizedonnitrocellulose As awidelyusedprotein andpolysaccharidemarkerforelectron microscopy, Gold, Colloidal Each kitissufficient for200applications(smallblotson8x10cmPAGE gels). stained protein bands,whichare easilydestained(reversed) withanEDTA solution. due tostrong chargeinteractions.ApplicationofactivatedR-PROBproduces lavender- Coomassie stains.Manyotherstainsproduce highbackgrounds onnylonmembranes and PVDFmembranes,aswellPAGE gelswith sensitivitycomparabletothatof unique protein detectionproduct forvisualizationofproteins onnylon,nitrocellulose, The ReversibleProtein DetectionKitforMembranesandPolyacrylamideGels isa Membranes andPolyacrylamideGels Reversible Protein DetectionKitfor water washes,facilitatingsubsequentimmunologicaldetection. blots), aswellcelluloseacetatemembranes.PonceauSstainiseasilyreversed with (5 minute)stainingofprotein bandsonnitrocellulose orPVDFmembranes(Western Ponceau SSolutionisaready-to-use, reversible stainingsolutiondesignedforrapid Ponceau SSolution rdc oe ecito Size Size O 9755 Description Product Code 50755 Description Product Code rdc oe ecito Size Size R-PROB Description Product Code Size P 7170 Description Product Code P 3504 Description Product Code i e 25 g 500ml Oil RedO Gold, Colloidal eesbePoenDtcinKt1kit 1L Reversible Protein DetectionKit 10 g Ponceau SSolution Ponceau S 100 g 100 g 50 g (w/v) sulfosalicylicacid.SuitableforSDS-PAGE, non-denaturingPAGE, andIEF. zation reagent. 1xfixingsolutioncontains12%(w/v)trichloraceticacidand3.5% Fixing solutionprevents diffusion orlossofproteins priortostainingwithavisuali- Fixing Solution,5xconcentrate fats, andsterols. Sudan BlackBisalipochrome (fatsolubledye)usedtostainphospholipids,neutral Sudan BlackB and buffer. light. Stainingsolutionsare commonlymadebydissolvingthedyeinformamide Typically, PAGE gelsare stainedinthedarkanddestainedthrough exposure to RNA bandsappearbluish-purple,DNAblue,andproteins appearred. Stains-All isuniquelysuitablefordifferential stainingofnucleicacidsandproteins. Stains-All ELECTROPHORESIS rdc oe ecito Size Size F 7264 Description Product Code Size 86015 Description Product Code E 9379 Description Product Code iigSlto,5 ocnrt 500ml 25g Fixing Solution,5xconcentrate 1g Sudan BlackB Stains-All 100 g 5 g 1 L

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Protein Analysis 113 Electrophoresis