• Growa2mlculture,24hr.at30oCinselectivemedia
• Whencultureisready,useittoinoculateabout55ml(50mlplus5forO.D.600reADIngs)ofselectivemedia.Growtomid-logarithmicphase(O.D.600isabout1).Thisusuallytakes10hours(for1mlofthe24hr.cultureinto50mlmedia).Itishelpfultoinoculate2differentflasks--onewith1mlofthe2mlculture,theotherwith0.5mlofthe2mlculture
• Spinat2750Xg.for10min.at30oC
• ResUSPendin100mlpre-warmedYPDsothatO.D.600=0.5(50mlofO.D.=1cultureinto100mltotalYPD)
• Ifappropriate,after1hradd2.5uMalphafactor(421ulofstock(1mg/ml)to100mlofculture)(wewantitpresentduringthelasthourofgrowth)
• Wait1doublingperiod(about2hours)untilO.D.600equalsabout1
• Add1mlofCOLD1MNaN3to100mlculture(wewant10mMtotalconc.)
• Spindown3x109cells(about100O.D.equivalents(100mlofOD=1):2750Xg.,10min.,30oCinlabclinicalcentrifuge
• Wash1xwith10mlSKbufferin15mlfalcontubes(seerecipebelow).
• Resuspendin10mlSKwith1mgzymolyaseand28.8mMBeta-mercaptoethanoltomakeSK:thawzymolyase(keptat5mg/mlstockinSKat-20oC)atroomtemp,thenspin1minat14,000rpm.Takesupe.--for4gradients,use900ulofsupe,90ulofbeta-mercaptoethanolandbringto45mlwithSK(thisrecipeshouldbeadjustedfortheappropriatenumberofgradients--wewanttoconservezymolyase--it"sexpensive)
• Shakegently,45min,60rpm,30oC

FROMHEREONOUTKEEPEVERYTHINGAT4oC

• Centrifuge500xg,10min.4oC.
• Wash1xwith2mlcoldSKbuffer
• Washagainwith2mllysisbufferC(Seerecipebelow)
• Resuspendin1mllysisbufferCandtransfertoglassP-Etube(potter-elvejhem--homogenizertube)
• Disruptwith25strokesofamotorizedhomogenizeronice(goentirelyinandoutofliquid25X)
• TransfertoEppendorftube,centrifugetwice(spinoncethenre-spinsup)at500Xg.for10min.,4oC
• Remove100ulofsupas"input."add100ul2XSDS-PAGEsamplebuffer.Putin100oCheatblockfor10min.,thentransferto-20oC.
• Add650ulofthesupto606mg(0.606g)sucroseinaultracentrifugetube(thismakesa70%solution)--usetweezerstohandleultracentrifugetubes,add"flea"(thetinystirbar).Putabunchoftubesinasmallbeakerandputthebeakeronastirplateinthecoldroom.leaveat4ountildissolved(aroundanhour)
• Removefleabytakingabigstirbarandputtingitagainsttheoutsideofthetube,movingitupwards.Gentlyoverlaywith1mlcoldsucrosesolutionsof60%,50%,40%,30%
• Balancetubeswith30%sucrosesolution(weighthemtoconfirmbalance)
• Spininswingingbucketcentrifugefor16hr(cangolonger)at190,000xg(37500rpm)inaSW55Tirotor
• Havemicrofugetubespre-labelledwith100ul4XSDSPAGEsamplebuffer
• Afterspinisdone,collect16samplesof300uleachinto100ulof4xSDS-PAGEsamplebuffer.
• Putat100oCfor10min,thenfreezeat-20oC--canstorehereindefinitely.

RUNNINGGEL

• Pre-warmsamplestoabout37oCforabout20-25min.Vortex,spinat14,000for1min.
• Pourgel:1.5mmthick,8%SDS-PAGEgelwithalongstackinggel--about1/2ofthegelshouldbeseparating,and1/2stacking.Alsousea15wellcomb
• Load20ulofsup
• Runat50voltsforabout4hr.
• Transferfor2hr(or1hr30min)at100V

SOLUTIONS

S/K(100ml):

1.2Msorbitol(21.84g/100ml)0.1MKPO4pH7.5(9.5gdibasic(fw=268)1.99gmonobasic(fw=136)

filtersterilize!

zymolyasebuffer:

toS/Kbufferadd:2ul/mlofbetamercaptoethanol20ul/mlofzymolyase(stocksolution5mg/mlinS/K)

spinbeforeusing!

LysisbufferC:

20mMTEApH80.8Msucrose1mMEDTA1mMDTT(addfresh)1mMAEBSF(addfresh)10ug/mlleupeptin(addfresh)10ug/mlpepstatin(addfresh)10ug/mlbenzamidine(addfresh)

mixfirst3ingredientsandfiltersterilize

Sucrose:

60gsucrose+5ml200mMTEA+H2Oto100mlfor60%solutionalsoprepare50%,40%,30%(allalsodissolvedinTEA)