YeastIFwithMethanol/AcetoneDehydration
DavidAmberg
NotethisprotocolisamodifiedversionoftheprotocolfromMarkRoseintheCSHYeastGeneticsCourseManual.
ThisistheprotocoltousewiththeBotsteinanti-actinrabbitantibodies.
- Growcellsattheappropriatetemperatureto5x10E6in5mlsYPD.Add0.5mls37%formaldehyde(bestgrade)andincubateontherolleratsametemp.for10min.
- Spindowncells2Kx3min.andresUSPendin5mls40mMKPO4pH6.5/500uMMgCl2+0.5mlsformaldehyde.Incubate1hrat30¡C(orprevioustemp).
- Washthecellstwotimesinthepreviousbuffer(noformaldehyde)andonceinthesamebuffercontaining1.2Msorbitol.Beverygentlewiththecells!Resuspendin0.5mlssorbitolbuffer.Thecellscanbestoredatthispointovernightat4¡C.
- Zymolyasetreatthecellswith30ul10mg/mlZymolyase(100T)at30¡Cforanywherebetween10-30minormore.Examinecellsonaphasemicroscope.Whencellsaredarkandmishapen,youhavegonewaytoofar,ifbrightandrefractiletheyprobablyneedtobeincubatedlonger.Iftheylookgoodbutaredullgreytheyarejuuuussstttright.Isuggestthisbedoneasatimecourseasitisthesinglemostvariablepartoftheprocedureandunfortunatelyitisthemostcriticalaswell.
- Washthecellsonceinsorbitolbufferandsuspendin100-500ulofthesame.BEVERYVERYGENTLE!Placeonice.
- Coatthewellsoftefloncoatedslideswith0.1%polylysine(>400,000MW)inwaterfor10minatRT.Spinthissolutionfor10mininamicrofugeimmedietlybeforeuseandstayawayfromthebottom.Ingeneraldohighspeedspinsofallsolutionsthatgoonthewellsrightbeforetheyareused.Incubatetheslidesinamoistchamber.
- Washthewells4-5timeswithcleanspunwateranddry.Thesecanbepreparedinadvanceifkeptdustfree.
- Spot20ulcellsuspensiononthewellsandincubateatRTfor10min.Isuggestyoudoeachsampleinduplicate.Aspirateoffmostoftheliquidbutnotall(donotdrytheslides)andplunge,thisisimportant,plungetheslideintoacoplinjarthatissurroundedbydryicefor6min.thenplungeintoanothercoplinjarthatcontainsacetoneandisalsosurroundedbydryicefor30sec.Itisimportantthatthejarsandcontentsaredowntotemperaturesoplacethemonthedryiceanhourortwobeforethisstep.Changeyourmethanolandacetonefrequentlyanddonotusesharpiestomarkyourslides.
- Whenyouremovetheslidefromtheacetoneimmediatlyplaceagainstaslanted,flat,warm,cleansurfacesothattheacetoneevaporateswithoutthecreationofcondensation.Iliketousethetopofawarmwaterbath.Itcanhelptowickawaytheexcessacetonefromthebottomedgewithakimwipeastheslidedries.Thisprocessshouldonlytakeafewsecondsandyoushouldbewearingglovesasfingergreasewillmakeamessofthings.Ialsowashthetalcumoffthegloves.
- BlockthecellsinPBSpH7.4/0.5%BSA/0.5%ovalbumin.Highspeedspinthissolution.Sometimestheinclusionof0.5%Tween20canhelpthespecificityoftheantibodyandshouldbeincludedinthisblocksolution.Beforewarnedthatthetweenreducesthehydrophobicityoftheteflonandsamplemixingcanoccur.Ifindthatifyououtlinethewellswithasharpyaftertheacetonestepyoucanpreventmixing.Incubatefor15minatRT.Thecellscanbeincubatedforovernightatthisstep.
- Incubatethecellsinblockcontainingantibodyattheappropriatedilution(determineexperimentally).Adilutionseriesof1:100to1:10,000shouldcoverit.IncubateatRTfor1hrorlonger,dependingontheantibody.Sometimesanovernightincubationishelpful.Jon"santi-actinGuineapigantibodiesfromanimal#1arebestat1:6,000,#2and#3arebestat1:2,000.
- Washthecells4x5minwiththeblocksolution.Sometimeslongerincubationtimesmaylowerthebackgroundbutthisisusuallysufficient.
- Incubateinsecondaryantibodyconjugatedilutedinblocksolutionfor1hratRT.Youmayneedtodeterminetheconcentrationbestforyoursecondaryantibodyaswell.TheCappelanti-GuineaPig-FITCisbestat1:800-1:1,000.
- Washasbefore.Aspiratemostoflastwashoffthecellsbutdonotdryandmountimmediatly.Mountbysloppingthemountsolutionovertheslide(nobubbles)andgraduallylayingthecoverslipdownbythelongaxis.Laypapertowelsovertheslideandsqueezeouttheexcessmount.Whileholdingtheslidedownwiththefingersofonehand,cleantheslidewithakimwipe.Finallysealtheedgesoftheslidewithnailpolish.IfindthatSallyHansen"s"HARDASNAILS"worksbest.Allowtodryandstoreat-20¡Cuntilyouarereadytoviewtheresults.Beforeyouplacetheslideonthescope,completelycleananyresidualmountfromtheslide,thisstuffisbadfortheobjectives.
Reagants
- Zymolyase
- Usethegoodstuff100T.Dissolveinthesorbitolcontainingphosphatebufferdescribedintheprotocol,spinfor10mininamicrofuge,removetoafreshtubeandquickfreezeonliquidnitrogen.Storeat-80¡C.Donotrepeatedlyfreezeandthaw,ifyoudoalwaysquickfreeze.
- Polylysine
- Buythegoodstufffromsigma,>400,000MW.Dissolveasa1%stocksolutioningoodwater.Quickfreezeinaliqoutesandstoreat-80¡C.Sameappliesasforzymolyase.
- DAPI
- Stocksolutionshouldbeat1mg/mlinwaterandstoredat-20¡C.
- Mount
- Dissolve100mgp-phenylenediaminein10mlsPBS,adjustthepHtoabove8.0with0.5MNaCarbonatebuffer(pH9.0)andbringthevolumeto100mlswithglycerol.AddDapito50ng/ml.Mixthoroughlyandstoreat-20¡C.Itturnsbrownwhenitisbad.
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