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本帖最后由 细胞海洋 于 2011-3-12 17:58 编辑
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( x7 X* M0 {; n0 l* |Introduction 1
/ w: K' o* D* _6 ^Western Blotting Overview 2-3: L# Z2 w' q+ g
Step 1 – SDS-PAGE 3 ]+ A6 W+ ^7 `" l" Z9 @
Thermo Scientifc Precise Protein Gels 4 " r4 L+ g+ h- X9 C9 m3 ?
Molecular Weight Markers 5-6/ T$ ]3 l0 @1 x9 a
Step 2 – Electro-Transfer
" Y6 ` {7 t+ N9 m( T" v Thermo Scientifc Pierce Fast Transfer System 7
^( Q. {4 g/ Q4 T. K Transfer Buffers 8 4 d2 k1 z$ f7 h- S8 W9 r
Filter Paper for Blotting 8 9 R/ d( j$ V2 K) ~) G9 y, _
PVDF and Nitrocellulose Membranes 9
* F, ]9 @( |9 D2 v$ b' i$ H! v4 h. _ Thermo Scientifc Pierce Protein Stains 9
: M! W' F7 O' b9 x5 Q for Membranes
- W8 C% U7 |0 R7 B, b Antibody Extender Solution NC 10
$ e3 D( B7 F0 e5 g9 b4 X Western Blot Signal Enhancer 11
4 @) Y4 O O* J2 RStep 3 – Blocking " l3 i& @/ J" Z* H
Introduction 12 0 `8 j5 M# e; }; f' m( ?
Blocking of Nonspecifc Binding Sites on 12 ; B0 Z8 U o, U. P' z
Transfer Membranes
$ h+ b$ B( ~6 J3 @ Blocking Buffer Optimization 12-13 , N( n2 b' ]9 ^. o/ }' a
Blocking Buffers 13-16
5 N+ `/ ^/ \& b, ?0 s# E; `Step 4 – Formulate Wash Buffers 2 t- i0 b# C$ R! w+ J% x
Washing the Membrane 17
/ ^; r* A- O8 x6 g4 v Wash Buffers 170 g/ V8 o) G2 K7 |: p+ C; `
Step 5 – Detection Reagents
& H& |% f7 C+ E( u% |! |5 k g Validated Primary Antibodies 18-19 ; k# b( _% E: q5 f) d8 u! v
Affnity-purifed Antibodies 20 , V- g& B9 F* r
Stabilized HRP Conjugates 21
; Y9 Y) H3 p$ r8 d6 }7 n Thermo Scientifc DyLight 22
4 o# ~9 E! K' E6 J' z$ k Fluor Conjugates % n- M/ E/ F8 w* T
Conjugate Stabilizer Solutions 22
# d7 j1 A- o( K) n4 a Thermo Scientifc DyLight-labeled Highly 23 4 q3 |$ E, u# Y" A
Cross-Adsorbed Secondary Antibodies
# ]% }8 Y+ N- _6 x: y* w& I Antibody Storage and Stabilizer Solutions 24 3 D) G# G5 d5 S$ d& K
Secondary Antibody Ordering Table 25-26
: W& D2 r) g. `' }) K+ h0 G: Z Thermo Scientifc Clean-Blot IP 28-29
; B9 m2 ]* W. l$ ]; ] Detection Reagents
! D1 K+ V' x$ i* c Protein A, G, A/G and L Conjugates 30 8 k, X" k& o6 |3 D4 \4 c
Thermo Scientifc NeutrAvidin, Streptavidin 31-33 $ M9 R( w! s! R0 G
and Avidin Conjugates
T* X, b+ m) i0 m7 H, b! kStep 6 – Enzyme Substrates $ [* N) j& k0 i
Chromogenic Substrates 34-35 M' f5 y; V3 g( H
Thermo Scientifc Chemiluminescent Substrates 36-43
6 k9 t8 H% l# S9 p Pierce ECL Substrate 37 2 u7 h5 l+ B! L! i) L; K) m
SuperSignal Chemiluminescent 38-42 ; J. w$ i; t" l8 i* g( o- T
Substrates and Kits
9 j$ P, i8 f0 D) i9 Y# d w& i Lumi-Phos™/ S2 C Z' L, J
Chemiluminescent Substrate 43 , U; H0 l. `/ L- J
Quick Reference Substrate Guide 43
/ u4 _" g9 ?7 v2 qFast Western Blotting Kit 44
9 H( N2 T( E# I7 \9 bSpecialized Western Blotting Kits 45-48
. }" |3 `: p2 s7 G: c# m( { Thermo SuperSignal West Pico HisProbe Kit 45 7 @) q6 {; E {
Pierce O-GLcNAc Western Blotting Detection Kit 46
* _% A: n& y6 L- O+ { Thermo Scientifc DyLight 549/649 46-47
m% q' D0 r8 o/ r1 ?, \6 R Western Blotting Kit
/ C+ ^/ d8 T' L) ]5 h Thermo Scientifc DyLight 680/800 Near Infrared 47
/ I3 D& B# [! [# Z3 O& B Western Blotting Kit
( r2 {- }. { @( M' [1 B Thermo Scientifc Active GTPase Pull-Down 48
5 v& S2 T) i) M; @8 `$ L and Detection Kits 2 F. `' a% d3 f% ?* H1 {
Far-Western Blotting 49 / I% ]4 f# X+ d; b2 c2 l" m
In-Gel Western Detection 50-51
" K+ t6 K4 _1 C# h Thermo Scientifc Pierce In-Gel 51 7 a# z% a. ?, r* [
Chemiluminescent Detection Kits
& Y- q. ^' }% V- r, WStep 7 – Film , J `7 l, I3 u: E8 Q
Thermo Scientifc CL-XPosure Film 52, H8 F' A8 }* P3 q# u5 @ ?
Step 8 – Stripping Buffer 0 n' B, l; ?0 X1 d
Optimizing the Signal-to-Noise Ratio 53-58
+ X7 c3 Z2 K1 h' x Protocol for Stripping an Immunoblot 54
6 f# a7 }4 t; Q1 Y+ g' N7 ? Thermo Scientifc Restore 55
, F+ F1 U: j J& ? Western Blot Stripping Buffers
5 a- J3 r9 J2 s2 Y8 S! @7 L, M3 G Thermo Scientifc Restore PLUS 56
) L! G% v1 d4 o' w# ^ Western Blot Stripping Buffers ; X, l5 K6 k" R1 m7 ]
Thermo Scientifc Pierce 57-58 3 e$ w& f$ ^3 z5 B' m
Background Eliminator# P" @9 T: q2 Y
Troubleshooting Guide 59-69 $ S4 X7 _) O* G
Blotting with Chemiluminescence 59
+ v+ N; P' q# U1 [' k4 Y% B; e: S7 w& Q Optimizing Antibody Concentration 60-63
# [, P; Z- J. U3 t+ O( s; F Problem Guide 64-67
" H, v8 n1 `1 m+ B7 W, I" c Full-Length Western Blotting Protocol Using 68-69 8 P a$ j4 p6 X6 S5 s3 B1 A& q
Chemiluminescent Substrates
: E% n8 h# m/ P2 nRecommended Reading 700 e( n% n3 K. z
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发表于 2011-3-12 09:56
