Materials:

Overlay/Rehydrationbuffer:

125mMTris-HCl,pH6.81mMEDTA0.1%SDS1mM2-b-mercaptoethanol30%glycerolsomeBromophenolblueFor1ml,youwillneed0.35mlof2Xbuffer0.30mlH200.30mlglycerol0.05mlBPB

Enzymebuffer:

125mMTris-HCl,pH6.81mMEDTA0.1%SDS1mM2-mercaptoethanol10%glycerolsomephenolredFor1ml,youwillneed0.45mlof2Xbuffer0.30mlH2O0.10mlglycerol0.15mlphenolred

Thebestwaytomakealloftheseistohaveastockthatdoesnotcontainmercaptoethanolandtomakeupwhatyouneedforonedayeachtimeyoudothis.Then,whenyouaredone,throwawaytheexcesscontainingthemercaptoethanol.Otherwisethereisnowaytoknowwhetherthereistoomuch,ortoolittlemercaptoethanolinthesolutions.Bothextremesareaproblem.IfyouhavetoolittleME,theproteinwon"tcomeoutofthegel.Ifyouhavetoomuch,theV8won"twork.Themolarityofneatmercaptoethanolisapproximately14.1microliterofMEper14mlofsolutiongivesa1mmolarsolution.

Enzymestocksolution:

500micrograms/ml(or500nanograms/microliter)V8-proteasein125mMTris-HCl,pH6.8,and1mMEDTA.ThisisinTilo"sbox3,intheeastForma,#1.

Generally,ifyouaren"tsurehowmuchenzymetouse,try10,50and100nanogramsofV8protease.IfV8doesn"tworkforyourprotein,trypapainorchymotrypsin.JanBussoncehadtouse1microgramofV8togetgooddigestion.Therearenorulesandeveryproteinisdifferent.WealwaystryV8first.

Youneedasetofworkingsolutionsthatwillgiveyouthedesiredweightofproteasein10microliters.forexample:

for10ng,use1microliterstockV8in500microlitersenzymebuffer.for25ng,use1microliterstockV8in200microlitersenzymebuffer.for50ng,use5microlitersstockV8in500microlitersenzymebuffer.

Thosefamiliarwithdifferentialequationsoranalyticgeometrycancalculateotheramounts.

Ingeneralshortgelsgivethebestlookingpatterns.Rememberthatthebottomofthestackinggelshouldbe30mmfromtheupperendofthenotchedplate,not23mm.Excisethegelpiece.Theprepgelshouldbeunfixed,unPPO"edandhavebeendriedafterrunning.ItisOKtosoakthegelinH20priortodrying.Scrapeoffbackingpaperandtrimthepiecesto3mm(whichisthesizeofthe28-toothcombwells).Rehydratethegelpiecesin100microlitersofrehydration/overlaybufferfor1-2min.Donotover-hydrate.Thepieceswillbecometoofragileandwillfragment.Placethegelpiecesinthewellsofthegel,usingtheteflontampertopushthemdowntothebottom.Add10microlitersoftheRehydration/Overlaybuffer.Thiswillsealanyspacebetweenthegelpiecesandthewells.Addthedesiredamountsofprotease(10,50,200ng,orwhateverworksforyourprotein)in10microlitersofenzymebuffer.Theproteaseinevitablyleaksfromonewelltoanother.Therefore,itisbesttogroupthesamplessuchthatallthataretobedigestedwith10ngofproteasearenexttoeachother,allthataretobedigestedwith50ngarenexttoeachother,andseparatedfromthosegettingdigestedwith10,andsoforth.Startelectrophoresisasusual.Whenthedyefrontis3to4mmabovethetopoftheresolvinggel,turnoffthepowerforexactly30min.Thenturnitonagain.Whenthedyeiscompletelyintheseparationgel,turnupthecurrenttowhateveryouwouldnormallyuseforthekindofgelyouarerunningandproceedasusual.