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Western Blotting

Preparation of Buffer and Reagent

(1) 10x Running Buffer: (2)10x TBS Buffer

Tris Base 30.4g Tris Base 24.2gGlycine 144.2g NaCl 80g

SS 10.0g a!! !!"2# $% 1& an! a!'us$

a!! !!"2# $% 1& "*.+

(3) Transfer Buffer: (4) 1, TBST (-as Buffer)

Tris Base (2/) 3.03g 10x TBS 100l

Glycine (12) 14.4g !!"2# 00l

!!"2# 800l Teen20 1l

e$an%l 200l

(/) /x laeili &%a!ing Buffer

(5) /0l 1 Tris"Cl ("+.8)

*.88g Tris"Cl6 a!! !!"2# $% a7%u$ /0l6 a!'us$ "+.8

(55) 1 Bl%%9en%l Blue

0.1g Bl%%9en%l Blue6 a!! !!"2# $% 10l

(555) 10l &aeili &%a!ing Buffer

3.00 l 1 Tris"Cl("+.8)

/.00 l Glycer%l

1.2/ l erca9$%e$an%l 0.2/ l 1 Bl%%9en%l Blue

1.00g SS

Protocol: Electrophoresis and blotting

1. ix sa9le (/0100ug) Cell lysa$e %r $issue lysa$e i$ /x &%a!ing

Buffer an! 7%il f%r / inu$es. nuse! sa9les us$ 7e s$%re! a$ ;20C

2. a

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$% $e ins$ruc$i%n %f ,cell 55 Bl%$ %!ule. Te c%n!i$i%n f%r $ransfer is

2/= c%ns$an$ f%r 1./ %urs.

Blocking the membrane

1. Bl%c< n%ns9ecific 7in!ing si$es 7y iersing $e e7rane in / n%n

fa$ !rie! il< in TBST 7uffer ( s%e$ie i$ nee!s s9ecial seru $% re!uce

7ac

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/. rain %ff excess !e$ec$i%n reagen$ 7y %l!ing $e e7rane gen$ly in

f%rce9s an! $%ucing $e e!ge agains$ a $issue. lace $e 7l%$s 9r%$ein

si!e !%n %n $% a fres see$ %f Sara-ra96 ra9 u9 $e 7l%$s an!

gen$ly s%%$ %u$ any air 7u77les.

+. lace $e ra99e! 7l%$s6 9r%$ein si!e u9 in an xray fil casse$$e6 9lace asee$ %f au$%ra!i%gra9y fil ("y9erfil DC&) %n $e $%9 %f e7rane6

ex9%se f%r sec%n!s (!e9en!s).