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1、BufferRecipesNameDescriptionRecipeBalancedSalt50mMTris,pH7.5150mMNaClFor1L:-6.05gTris-800mldH2O-8.67gNaCl-pHto7.5-Diluteto1LDMEM(10X)-Madefrompowder-UsedformakingcollagenmatricesforexplantculturesForadetaileddescriptionseemediaformulations-80mlofdH2O-AddDMEMpowder(Sigma#D7777-1L)
2、-Mixthoroughly-pHto1.8with10MHCl(~20drops)-Mixuntilcompletelydissolved-Bringto100mlwithdH2O-Syringefilter+aliquotLeibovitz'sL-15MediumInvitrogen#41300-039(powder)11415-064(liquid)Variousvitamins,saltsandaminoacidsForadetaileddescriptionseemediaformulationsFor5Lfrompowder:-Rinseou
3、tglassbottleswithwater(nosoap)-Autoclave-Ina5Lbeaker,add4Ldistilledwater-Dissolve5packsofpowder-pHto7.6withHCl-Bringto5Lwithdistilledwater-FiltersterilizeintoautoclavedbottlesundersterileconditionsPBS(Phosphatebufferedsaline)pH7.4137mMNaCl2.7mMKCl4.3mMNa2HPO41.4mMKH2PO4For1Lof10x
4、:-80gNaCl-2gKCl-14.4Na2HPO4-2.4gKH2PO4-pHto7.4NP-40lysisbuffer10mMTris,pH7.5150mMNaCl1%NP-40For50ml:-500mlof1MTris,pH7.5-40.75mlofdH2O-5ml10%NP-40-3.75mlof2MNaClRIPAlysisbuffer(RadioImmunoPrecipitationAssaylysisbuffer)50mMTris,pH7.5or10mMPhosphate,pH7.5*150mMNaClDetergents:1%NP-4
5、00.1%SDS0.5%DeoxycholateFor50ml:-2.5mlof1MTris,pH7.5or2.5mlof200mMNaP,pH7.5*-35.75ofdH2O-3.75mlof2MNaCl-5mlof10%NP-40-2.5mlof10%sodiumdeoxycholate-500μlof10%SDS-Bringto50ml*Phosphateisausefulbufferbecauseitinhibitsphosphatases,howeveritwillprecipitateinthepresenceofcalciumormanga
6、nese.SDS-PAGERunningBuffer(10X)Whendilutedto1X:25mMTris1%SDS200mMGlycineFor1Lof10x:-30.3gTRIS-10gSDS-144gGlycine200mMSodiumPhosphateFor50ml:-1.41gSodiumPhosphate-40mldH2O-pHto7.4-Bringto50ml50xTAE(TrisacetateEDTA)Whendilutedto1X:0.04MTris-Acetate0.001MEDTAFor500ml:-121gTrisBase-2
7、8.6mlglacialaceticacid-9.3gEDTA(or50mlof0.5MEDTA)-dH2Oto500ml1MTrisFor50ml:-6.06gTris-40mldH2O-pHto7.5with>5ml10MHCl-Bringto50mlTBS(Trisbufferedsaline)50mMTris137mMNaCl2.7mMKClFor1L:-800mldH2O-16gNaCl(or68.5mlof2MNaCl)-0.2gKCl-50mlof1MTris,pH7.5(seeabove)-Bringto1LwithdH2OWestern
8、TransferBuffer(10X)Whendilutedto1X:20mMT
