Basic Protein Chemistry Techniques

CoomassieBlueStain:(forgels)1)Combine225mlMethanolwith225mlddH2O.2)Add0.5gramsofCoomassieBlue.3)Justbeforeuse,add50mlaceticacidto10%finalconcentration.4)Stainfor2hours.Stainingtimecanbeshortenedbymicrowaving;~50secondsatmediumpower.5)Afterstaining,pouroffthestainintoabottlemarkedUsedStain.Staincanbere-usedonce,butfresh10%aceticacidmustbeadded).CoomassieBlueDestain:(forgels)1)Combine25mlofMethanolwith437.5mlddH2O.2)Justbeforeuse,add37.5mlaceticacidto7.5%finalconcentration.3)Destain,plussponges(piecesoffoampacking)foracoupleofhours.Itmaybenecessarytousemorethanonechangeofdestain.Microwavingcanbeusedinthisstagealso,butthedetailshavenotbeendetermined.CoomassieBlueStain:(formembranes)1)Combine50mlofMethanolwith50mlofddH2O.2)Add0.1gofCoomassieBlue.3)Stainbypouringasufficientamounttocoverthemembraneinapetridish.4)Washthefluidgentlebackandforthoverthemembrane.Allowittobeimmersedforonetotwominutes.5)Disposeofstaininamannersimilartothatforthegelstain.DialysisTubingPrep1)Cuttubingtoappropriatelengthsandplacetheminalargebeaker.2)Addenough5%NaHCO₃(add50gramsperliterofddH2O)tocoverthetubing.3)Bringtoarollingboilandallowittoboilfor15minutes.Thetubingwillfloatsoplaceabeakerofthenextsmallersizeontopofthetubing.4)RinsethetubingwithanexcessofddH2O.5)Soakthetubingin2mMEDTA(add10ml0.2Mto1literddH2O)foronehour.6)RinseagainwithanexcessofddH2O.7)Storein0.05%Azide(add0.005mlperliterofddH2O).ToPourStackingGel...0.4mlsAcrylamide0.4mls10xStackingBuffer40uls10%SDS3.12mlsddH202ulsTemed34ulsAPS

HowtoMake15%Mini-gels**Important**DuetothefactthatAcrylamideisbeingused,itisextremelyimportanttowearglovesthroughoutthisprocedure.1)Measureout5.152-mlddH2Ointoasterile15-ml-tube.2)Add4.58-mlAcrylamide.3)Nextadd1.1-ml10xSeperatingBuffer.4)Thenadd110-ml10%SDS.5)Continuebyadding56-mlAPS.6)Finally,add5.6-mlTemed.(Note:Temedisthepolymerizingagentsothefollowingstepsmustbedonefairlyquickly.)7)Useadisposablepipet,homogenizethesolutionbeingcarefulnottocauseanybubbles.8)Thenusethesamepipettofilltheglassleavingabout1inchatthetop.9)Finally,add0.5inchofH2O-saturatedbutanolontopofthegel.Note:Leavetheunusedportionofthesolutioninthe15-mltube.Usethistodetermineifyourgelisreadytouse(whenthetubeispolymerized,soisyourgel).