SolutionPreparation
Lysisbuffer:
0.15MNaCl,5mMEDTA,pH8.0,1%TritonX100,10mMTris-
Cl,pH7.4.Justbeforeuse,add5mMDTT,0.1mMPMSFin
isopropanol,5mMε-aminocaproicacid.
2XSamplebuffer:
130mMTris-Cl,pH8.0,20%(v/v)glycerol,4.6%(w/v)SDS,
0.02%bromophenolblue,2%DTT.
PBS,pH7.4:
10mMNa2HPO4,1.8mMKH2PO4,50mMNaCl,2.7mMKCl
RIPAbuffer:
50mMTris-HClpH7.4,150mMNaCl,1mMEDTA,1%Triton
X100,1%Nadeoxycholate,0.1%SDS,1mMPMSF,1µg/mL
aprotinin,1µg/mLleupeptin.
Protocol
A.Preparationofcelllysates(fromT-25flask)
1.Collectconfluentcellsbytrypsinizationandspin.
2.Lysethepelletwith100µlLysisbufferonicefor10min
(use20µlLysisbuffer/500,000cells).
3.Spinat14,000rpm(16,000g)inamicrocentrifugetubefor
10minat4°C.
4.Transferthesupernatanttoanewtubeanddiscardthe
pellet.
5.DeterminetheproteinconcentrationbyBradfordassay.
6.Mix1volumeoflysate(0.5mgprotein/membrane)with1
volumeof2Xsamplebuffer.
7.Boilfor5minandcoolatroomtemperature(RT)for5min.
8.FlashspintobringdowncondensationpriortoloADInggel.
B.Preparationoftissuelysates
Alwayskeeplysateortissueoniceatalltimesduring
preparation.
1.Removetissues,andweigh1.5gramsofeachtissue.
2.Chopthetissueintosmallpieces,washtwicewithice-cold
PBS.
3.Transferchoppedtissueintogrinder,andadd5mlRIPA
buffer,homogenize20times.
4.Transferhomogenizedsolutioninto1.5-mlmicrocentrifuge
tube.Spinat14,000rpm(16,000g)for10minat4°C.
5.Carefullyremovethelipidonthesurfaceofthe
supernatant.Savesupernatantaswholetissuelysate,and
discardthepellet.
6.DeterminetheproteinconcentrationbyBradfordassay.
7.Adjustconcentrationto2.5mg/mlwithLysisbuffer.Aliquot
100µlpervial,andstoreat-80°C.
8.Forwesternblotting,add100µl2XSDSsamplebuffer,boil
for5minutes,andstoreat-20°C.