PDF电子书：Western Blotting Handbook and Troubleshooting Guide(version 2)

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# i; M1 A, O- G; b3 M% K. \, v( aIntroduction  1
1 ?( r& j* `) B, bWestern Blotting Overview  2-3  t  q: r6 r( T) R# w9 B, o- t
Step 1 – SDS-PAGE
  W  }& ^, c3 Q+ @9 l  Thermo Scientifc Precise Protein Gels  4
: d5 H) ~. [* I+ C! C6 x: b6 g  Molecular Weight Markers  5-6
% P9 y% t5 Q7 D9 I  V2 b; U! hStep 2 – Electro-Transfer $ s2 |# _& q! y) D
  Thermo Scientifc Pierce Fast Transfer System  7
$ Z3 V% l1 x) V# r/ J( i  Transfer Buffers  8 ; ?+ I0 K$ P& b, }8 V! @% h& A
  Filter Paper for Blotting  8 , P4 C* r) `/ \) }
  PVDF and Nitrocellulose Membranes  9 9 F/ t  K3 Q* c! Y3 B
  Thermo Scientifc Pierce Protein Stains   9  * O; z0 W8 \, Y6 i! @2 Y* `
   for Membranes   + r9 N5 Q. N& o8 J3 _. b2 s
  Antibody Extender Solution NC  10
2 [8 p7 a& G' ~" N) X" B% T1 t  Western Blot Signal Enhancer  11% G* m  A* e  D' p$ A  H5 n
Step 3 – Blocking
% ^# a; U1 Y) ?8 E  Introduction  12 + I& f+ a- I  u" i. S
  Blocking of Nonspecifc Binding Sites on  12 7 A# G& G* j$ @, L8 j
   Transfer Membranes   & u1 M) T" M& B: `; R
  Blocking Buffer Optimization  12-13
- d) ]% o4 a' B, V' x3 g9 j/ y1 |  Blocking Buffers  13-166 h* J+ S0 k) ]; w
Step 4 – Formulate Wash Buffers
& ~; G4 `/ D0 k6 e6 h5 P1 B: b# a  Washing the Membrane  17
& [: V; d4 i" t) }8 N& h  Wash Buffers  174 T! u! f: E  p; b9 O) `, p. M/ D& G
Step 5 – Detection Reagents
: p. k# [% S. ]5 g  Validated Primary Antibodies  18-19
2 }$ `- ]' z3 ^, p( o& l( L  Affnity-purifed Antibodies  20 - h" d& P, _% U: `6 }
  Stabilized HRP Conjugates  21 2 A& K7 @  x; r, P0 h* y7 {
  Thermo Scientifc DyLight   22
2 t& v. y* j: X# |, ?6 ?4 R- I! i5 W   Fluor Conjugates   0 D2 ]1 S! F9 j$ S
  Conjugate Stabilizer Solutions  22 6 C% F$ y/ ?! g, B' u/ d
  Thermo Scientifc DyLight-labeled Highly  23
# `  I% i; U9 T: S   Cross-Adsorbed Secondary Antibodies 3 }' Q( Z7 S* D& b2 V$ M. r
  Antibody Storage and Stabilizer Solutions  24
& Y& L, }  F8 |+ V; z6 ?3 _  Secondary Antibody Ordering Table  25-26  - B" Q1 b0 r/ x1 u" L& r
  Thermo Scientifc Clean-Blot IP  28-29 / c6 q% }1 A! W3 ~' u  X
   Detection Reagents   
  l( |! q7 Y. Y  Protein A, G, A/G and L Conjugates  30 , Y; @3 Y$ {) c5 L
  Thermo Scientifc NeutrAvidin, Streptavidin   31-33 : m* ~8 g+ W1 }) e# h  f2 t+ J
   and Avidin Conjugates
9 C0 K* `6 }0 C  m: z- U- hStep 6 – Enzyme Substrates
% \! l, Q" W' {1 |% WChromogenic Substrates  34-35
) f# \; O  K( X* R' W. Z. L) WThermo Scientifc Chemiluminescent Substrates  36-43
0 P/ f3 A0 `/ V0 d  Pierce ECL Substrate  37
# o2 b- m9 c/ ^" v6 i" m' v  SuperSignal Chemiluminescent   38-42 * }+ d, d) t% U0 _) D% ]/ ^
   Substrates and Kits   
& j! G. i( [& ]6 T  Lumi-Phos™
+ W7 P( L. Q5 T* L Chemiluminescent Substrate  43
4 N( v: w3 [( W+ \8 r  Quick Reference Substrate Guide  43
+ H* l7 v) V' e* u4 QFast Western Blotting Kit  44
! R; C" R" R, B* b8 CSpecialized Western Blotting Kits  45-48
2 ^3 ~- W! O  P" U4 O  Thermo SuperSignal West Pico HisProbe Kit  45
& g9 i, u: S. f7 x& S2 g+ F/ U5 o  Pierce O-GLcNAc Western Blotting Detection Kit  46
) [6 U0 S" Y1 j% s3 {  Thermo Scientifc DyLight 549/649  46-47  $ K9 t* r' f3 Q# ^9 }$ M1 y
   Western Blotting Kit   
3 Q  l6 n  u: b- s  v& c5 O2 r( e  Thermo Scientifc DyLight 680/800 Near Infrared  47 8 I* I9 b& _& C# V: K, d3 L
   Western Blotting Kit   
3 o! C9 {7 M5 C* V  Thermo Scientifc Active GTPase Pull-Down  48 4 v# F, J# L6 h$ _0 Z
   and Detection Kits   
5 g! D: u5 x/ `) [& x/ [Far-Western Blotting  49
2 D5 k: |1 M$ ?! v5 _$ a: L8 N9 WIn-Gel Western Detection  50-51 * J0 F/ c# Z9 {3 x  @, Q
  Thermo Scientifc Pierce In-Gel   51
( g( r" g+ P! O. {   Chemiluminescent Detection Kits# L7 D) L7 d6 Z
Step 7 – Film - B% M1 d/ n* g5 J1 S# [
  Thermo Scientifc CL-XPosure Film  52
6 @9 t6 \% J& P) `' |* BStep 8 – Stripping Buffer 5 w7 a) h: @, o: M
Optimizing the Signal-to-Noise Ratio  53-58 ' @* F: b4 k' B( ?* V+ h
  Protocol for Stripping an Immunoblot  54
% d7 [/ V$ ~+ @! m  U  \  Thermo Scientifc Restore  55
& X$ S- C- y5 k" @  y   Western Blot Stripping Buffers
# |8 J1 ^( A+ K% j0 e+ T  Thermo Scientifc Restore PLUS  56 ; z  A' k; E1 N
   Western Blot Stripping Buffers   Q# m' W8 Y1 g  h
  Thermo Scientifc Pierce   57-58 " l0 T" _: r$ _& W  a1 P4 Z
   Background Eliminator: k- ]( Q1 W5 e3 H8 }8 R
Troubleshooting Guide  59-69 % h# E+ _# W& ?
  Blotting with Chemiluminescence  59 + }* Q3 |3 Z8 W& e
  Optimizing Antibody Concentration  60-63 ! O/ T3 O# {3 X1 i% b" G1 ~
  Problem Guide  64-67 8 q# y( s* R- a# t
  Full-Length Western Blotting Protocol Using   68-69 8 f) j- j: R- D2 d  ^5 r( x
   Chemiluminescent Substrates
: b% c# d$ ^0 `  M+ `) W8 lRecommended Reading  701 }9 c7 V) M9 I' N! H0 C" h

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一枝独秀

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jiandong2001

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军医

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深海寂寞鱼

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sunyucheng

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daviddow

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mominglily

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