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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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" H5 r5 o7 H* C# z" [Introduction 1
/ ] M) U9 m* |$ r8 a6 ]; V$ nWestern Blotting Overview 2-33 R8 I* O" M' C. \2 d
Step 1 – SDS-PAGE
8 }3 i, ]: {: ^; L, F/ D' N Thermo Scientifc Precise Protein Gels 4 6 c/ E) q" N, u2 w
Molecular Weight Markers 5-6) \5 v& W* w5 `' ?& K! J9 V
Step 2 – Electro-Transfer
% i* T( k2 K' H( ~/ }0 { Thermo Scientifc Pierce Fast Transfer System 7
; ]# a9 P) ~. U; w' L% o4 w0 G Transfer Buffers 8
) i1 w6 G7 I. S5 C Filter Paper for Blotting 8
: [8 _3 Z9 D8 G8 r0 s& I PVDF and Nitrocellulose Membranes 9
9 f* o9 K/ x3 S5 X Thermo Scientifc Pierce Protein Stains 9 ! c! v4 t* ?! N
for Membranes & }- |4 {9 o9 l* i3 k; j5 K; w8 S
Antibody Extender Solution NC 10
p2 Q/ i# [8 a/ T' z2 U Western Blot Signal Enhancer 11
. x9 ^% ]( X3 W- B4 m% X- m# VStep 3 – Blocking 8 y4 B1 `/ d. p2 u
Introduction 12 ) d0 Z1 p( l& q. n/ |
Blocking of Nonspecifc Binding Sites on 12
3 X+ p5 G3 @7 r8 q# I Transfer Membranes $ _5 E1 @6 Q" ]; o, _
Blocking Buffer Optimization 12-13
. U4 ]3 F) [* k" V" [ Blocking Buffers 13-16
9 z/ }$ w1 y JStep 4 – Formulate Wash Buffers & x& R0 w7 T: d
Washing the Membrane 17
* F% G2 g: r1 p C- ^5 | Wash Buffers 17
# ^& p# D! r8 K" t) @Step 5 – Detection Reagents 5 [+ q" h8 U4 P8 F; `8 O
Validated Primary Antibodies 18-19
0 T7 U! ~0 X: v' L/ g: k Affnity-purifed Antibodies 20
, @3 n! i6 o: q+ J: i7 F6 F: i Stabilized HRP Conjugates 21
# }) x$ p J2 u) U% V Thermo Scientifc DyLight 22
8 C2 F% @4 t& t1 |0 A: Q Fluor Conjugates
7 }0 W3 q, M, k ^ Conjugate Stabilizer Solutions 22 5 _6 L) W6 ?$ l" W6 g/ D
Thermo Scientifc DyLight-labeled Highly 23
+ j0 Q; _0 n4 c Cross-Adsorbed Secondary Antibodies
9 B2 ~# r" g; u' W- j Antibody Storage and Stabilizer Solutions 24 # @; g, j: I/ z; ~0 @4 C0 G `
Secondary Antibody Ordering Table 25-26 % q' d9 K% y) n0 M6 U) o
Thermo Scientifc Clean-Blot IP 28-29 8 R6 F6 p1 b X
Detection Reagents ; t; O1 I7 c l: @7 S: I) @
Protein A, G, A/G and L Conjugates 30
2 G3 n J# h% e6 g( F0 w: n Thermo Scientifc NeutrAvidin, Streptavidin 31-33 % d! `( o. a8 D, `
and Avidin Conjugates
% }8 Q0 B: r( J1 G) hStep 6 – Enzyme Substrates
) S7 q& q/ N6 b: C9 C) n2 zChromogenic Substrates 34-35 ! O3 v$ V1 }7 E1 f
Thermo Scientifc Chemiluminescent Substrates 36-43 2 A8 U! }0 j7 j9 j8 J8 U' K8 }
Pierce ECL Substrate 37 4 h( K* h) S$ ^6 H7 n. B3 h8 _* ~
SuperSignal Chemiluminescent 38-42 8 i& ]+ C* n) O
Substrates and Kits 2 W" f- l' m2 M' I8 E
Lumi-Phos™
4 c F6 B/ T/ p1 q Chemiluminescent Substrate 43
7 W, C8 f4 F/ R! d7 } Quick Reference Substrate Guide 43 9 [' d+ F$ H% V& T
Fast Western Blotting Kit 44
# P1 b% x- N USpecialized Western Blotting Kits 45-48
# M+ {3 ?9 G. h& p Thermo SuperSignal West Pico HisProbe Kit 45
, k8 g& s- z y0 b9 R& O0 R Pierce O-GLcNAc Western Blotting Detection Kit 46 6 [* X3 m o& ~0 {% V! W; T5 z
Thermo Scientifc DyLight 549/649 46-47 ( o- U, E; L9 c' o% d
Western Blotting Kit
# {7 O; o$ F+ ]7 E% h5 ^ Thermo Scientifc DyLight 680/800 Near Infrared 47 & d3 e7 t2 _+ X& j9 G
Western Blotting Kit
6 D& ~+ j% g3 J& u Thermo Scientifc Active GTPase Pull-Down 48
5 `' e- t; _7 m8 x and Detection Kits
" T4 U* q/ S" e1 CFar-Western Blotting 49
7 T5 B& `& }* ]" o0 nIn-Gel Western Detection 50-51 8 \! ]4 j8 F% m) Q, v/ W( Q
Thermo Scientifc Pierce In-Gel 51
8 x# J& r' p: E( f9 r- L Chemiluminescent Detection Kits
1 e% v. `" j! t4 R+ A: T; xStep 7 – Film
/ N9 S( a( o$ v2 d8 M8 U$ Q+ k Thermo Scientifc CL-XPosure Film 52
' ^& I& Z* \& a7 qStep 8 – Stripping Buffer
9 t& m8 z# p7 l0 oOptimizing the Signal-to-Noise Ratio 53-58
8 ~. Z0 B Y2 b: F2 E1 d5 Y: A Protocol for Stripping an Immunoblot 54
! y% @+ L" @% K Thermo Scientifc Restore 55 # d+ ]) H7 q8 j9 u8 b- d
Western Blot Stripping Buffers 9 l( Y3 c' l$ a8 L U
Thermo Scientifc Restore PLUS 56
+ `' z) D& x3 D; ^ Western Blot Stripping Buffers
* w3 V) U8 B; E3 g' u4 z4 o Thermo Scientifc Pierce 57-58
* x% }# D. q+ J, ?; U Background Eliminator
* y$ k, F6 y& E/ f5 jTroubleshooting Guide 59-69 " ~. h5 y' }5 Z# e* K$ m0 k
Blotting with Chemiluminescence 59 . J& o, ^( g, p- S+ f
Optimizing Antibody Concentration 60-63
1 V4 H8 R) _! _" D4 k: ?7 L Problem Guide 64-67 z$ [& K9 ?" i9 e) \8 G
Full-Length Western Blotting Protocol Using 68-69 5 A3 o# `! |. W# s3 x8 u) z3 A4 Z; A
Chemiluminescent Substrates . n# s4 k& s* ?& U( f7 f
Recommended Reading 70
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发表于 2011-3-12 09:56
