|
- 积分
- 1343
- 威望
- 1343
- 包包
- 387
|
本帖最后由 细胞海洋 于 2011-3-12 17:58 编辑 " h8 Z ^0 t& Q# H* }' j
) U: v0 v3 ^( m/ dIntroduction 1
$ X; n' ^1 q% O6 v6 i: G U2 JWestern Blotting Overview 2-3
: t( m$ k! T# U. dStep 1 – SDS-PAGE
6 Z) Y: w6 e! D8 D1 ^+ s Thermo Scientifc Precise Protein Gels 4
. b: }8 [" M5 t2 X; D Molecular Weight Markers 5-6- A+ d9 G# m2 x" u: n1 v* d0 P
Step 2 – Electro-Transfer 2 N6 a# ?" q' b1 H: d
Thermo Scientifc Pierce Fast Transfer System 7
9 c$ z$ q& `7 H9 U" ~; M9 x* A Transfer Buffers 8
9 R* e0 y; m2 A/ Y$ I Filter Paper for Blotting 8
- z% R# P& C' } PVDF and Nitrocellulose Membranes 9 & T$ V5 e1 Z( T
Thermo Scientifc Pierce Protein Stains 9 9 [/ P; }/ Y; q0 z! g3 v+ R; z- |
for Membranes 2 f: |" S$ w( J% s f! x( E
Antibody Extender Solution NC 10 . _! L7 P9 V4 ~- P5 y; t( I: C; Z
Western Blot Signal Enhancer 11
0 H. N8 t' U2 [% zStep 3 – Blocking
. Q# k6 z. X$ f9 V- U1 h1 d+ f; ? Introduction 12
' P: Q( a5 K0 @; X( X( @: N8 [ Blocking of Nonspecifc Binding Sites on 12
" q" f. G( v! O5 Y5 U8 Z Transfer Membranes
% Q6 S3 D6 D9 M8 [ Blocking Buffer Optimization 12-13 ( X/ P+ Z: S: i6 ?3 ]
Blocking Buffers 13-16
& p0 O% S1 h9 j" ?4 u3 n/ e% RStep 4 – Formulate Wash Buffers # c& o1 @6 e- Z% a8 A }
Washing the Membrane 17
# R5 b/ ?) V/ h3 Z Wash Buffers 17' P% ~' C& N1 d- S* g
Step 5 – Detection Reagents
( A" b3 X: K% O Validated Primary Antibodies 18-19 # s+ B5 b7 v% z& b
Affnity-purifed Antibodies 20 6 @3 q" s2 }9 y' O
Stabilized HRP Conjugates 21
* F& w: N, r6 W1 I: q- f Thermo Scientifc DyLight 22 1 U5 ^) a, f) _6 f+ T
Fluor Conjugates
( T" R1 e6 ] J+ Z! X Conjugate Stabilizer Solutions 22 8 k8 b$ W4 e/ k& A" \
Thermo Scientifc DyLight-labeled Highly 23 ! R; _3 m) f2 J+ [4 j. }1 ?8 b5 g4 F
Cross-Adsorbed Secondary Antibodies # d- F7 j8 |+ R# e2 u' @
Antibody Storage and Stabilizer Solutions 24
3 s% P9 T2 o: n Secondary Antibody Ordering Table 25-26
, X. V9 c0 u9 u+ J# ?2 C Thermo Scientifc Clean-Blot IP 28-29 3 t$ s3 b4 z5 n" X/ h: ]9 x. r$ @
Detection Reagents
! E. k2 B z1 b6 b* ~2 Q. z Protein A, G, A/G and L Conjugates 30 * i$ K8 O% a" b8 l8 a/ x
Thermo Scientifc NeutrAvidin, Streptavidin 31-33 2 }% e3 R2 v4 h* G7 n
and Avidin Conjugates2 q5 V# i; o: {% A& r
Step 6 – Enzyme Substrates
4 F8 V6 B) ]4 y: E mChromogenic Substrates 34-35 ! k) t8 V! r0 L( Y# I7 S% |4 X+ X
Thermo Scientifc Chemiluminescent Substrates 36-43
( q% j/ l, O, C6 w Pierce ECL Substrate 37 % {. m. h7 V7 m, Y2 g
SuperSignal Chemiluminescent 38-42
5 C+ I1 [2 M4 l- f* R6 F Substrates and Kits
$ }6 z, j' f. Y; k; s Lumi-Phos™
' a# ^+ V3 |0 y( r6 M( o6 n Chemiluminescent Substrate 43
( ?# X) [7 Z" t% u: V. w Quick Reference Substrate Guide 43
+ c1 O1 Z1 G6 {- C2 `! n+ |Fast Western Blotting Kit 44
" p2 e2 \2 ^8 C6 u6 x% tSpecialized Western Blotting Kits 45-48 3 b# N3 w- i6 S+ R7 R0 P
Thermo SuperSignal West Pico HisProbe Kit 45 8 o/ p$ ^& B+ R2 u
Pierce O-GLcNAc Western Blotting Detection Kit 46 ( W: Y0 @/ o! ?4 I& L# f" N
Thermo Scientifc DyLight 549/649 46-47
* @6 ^4 g+ l8 `3 R9 R+ g! U Western Blotting Kit
) {' ]7 X" \6 W9 L! s Thermo Scientifc DyLight 680/800 Near Infrared 47
; j0 r6 ~/ `3 x8 S Western Blotting Kit % K) T: m, K1 e$ V& P
Thermo Scientifc Active GTPase Pull-Down 48
: h: I! _( w8 F- {1 H. g and Detection Kits + C- F$ ]; e$ l1 ]& {6 V
Far-Western Blotting 49
; f6 _* o: W- b5 W5 c& X5 |In-Gel Western Detection 50-51 6 F1 j$ W: @! L
Thermo Scientifc Pierce In-Gel 51
# w( X1 }3 q) g! I- _ Chemiluminescent Detection Kits- X# _+ }3 X6 W
Step 7 – Film
7 Q" L# d5 F; `1 [& R( K6 O Thermo Scientifc CL-XPosure Film 52' c1 |. F1 \! x9 R7 o
Step 8 – Stripping Buffer 4 h# M) d! P: ?3 b$ U
Optimizing the Signal-to-Noise Ratio 53-58
( H/ l5 S1 u$ d+ o6 K/ L Protocol for Stripping an Immunoblot 54
+ d7 n+ K# w/ M# _, V Thermo Scientifc Restore 55
, P. W6 T7 ^$ Y2 ?2 R Western Blot Stripping Buffers
; {3 `: s/ S2 ] Thermo Scientifc Restore PLUS 56
% D; l6 O; T2 [/ P; k, w Western Blot Stripping Buffers
. ?; [0 f3 d% R0 l Thermo Scientifc Pierce 57-58 ( q- v' u* R& C5 B6 r2 ^
Background Eliminator7 r& e9 B: N9 Z6 h
Troubleshooting Guide 59-69
0 q1 i& Y8 m J" p Blotting with Chemiluminescence 59 " M+ l$ y7 A' u3 b
Optimizing Antibody Concentration 60-63
9 D% d' C) k! g2 Z# v; ` Problem Guide 64-67 8 W1 `3 `/ Q/ h; v$ `2 \0 f
Full-Length Western Blotting Protocol Using 68-69 * `9 Y5 _. V) P5 E2 Y
Chemiluminescent Substrates ! B0 X4 B0 t( a, P
Recommended Reading 70
9 d r7 S: t1 `$ F, A4 P" X# t: u) T3 G
[hide][/hide] |
附件: 你需要登录才可以下载或查看附件。没有帐号？注册
-
总评分: 威望 + 5
包包 + 10
查看全部评分
|
发表于 2011-3-12 09:56
