(7.8)--SDS-PAGE and Western blot细胞生物学细胞生物学.pdf

Protocol#13SDSandWesternblot

Protocol#13

SDSandWesternblot

Introduction

Proteinsareextractedfromcellsandseparatedbysizeusingpolyacrylamidegel

electrophoresis(PAGE).Thismethodusesprecastgelswhicharequicker,saferandgive

betterresolutionandreproducibiltythatself-preparedgels.Recipesforself-preparedare

providedintheindex.Followingtransferoftheproteins,thenitrocellulosemembranewillbe

blocked,andprobedwithaprimaryantibody.Boundprimarywillthenbedetectedwith

HRP-conjugatedsecondaryantibodyandthisdetectedusingECLreagent.

Aims

YouwillbeprovidedwithHEK/COScellsthathavebeentransfectedwithfourconstructsin

differingamountsasshownbelow.Note,inallcases,thetotalamountofDNAusedforthe

transfectionis2.0µg.Workingingroupsoffour,taketwosampleseachandshareonegel

betweenthegroup.Note–eachgelkitholdstwogels.

SamplepP2X2pP2X2-EEantisensepcDNA3.1ratio

A–––2.0µgN/A

B2.0µg–––N/A

C–0.2µg–1.8µgN/A

D–1.0µg–1.0µgN/A

E–1.8µg–0.2µgN/A

F–0.2µg1.8µg–1:9

G–1.0µg1.0µg–1:1

H–1.8µg0.2µg–9:1

AandBarenegativecontrols

C,DandEarepositivecontrols

F,GandHareexperimentalclonesandshouldbecomparedtoC,DandErespectively

Timetable

Day1Preparesampl