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

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1 z  z8 n9 S9 \6 E# H9 zIntroduction  1, v- J; L) G( x) t
Western Blotting Overview  2-3
4 o7 z9 B- l9 L# nStep 1 – SDS-PAGE 5 ^- [8 M" P' w. A6 G1 i
  Thermo Scientifc Precise Protein Gels  4 + k; Q  ?* P# Q  I3 i) b- j
  Molecular Weight Markers  5-6
1 M3 X+ `% v' Y  X/ {4 VStep 2 – Electro-Transfer ' g2 D* m# C! s) M, L
  Thermo Scientifc Pierce Fast Transfer System  7 3 p& o: t! C- }! ^
  Transfer Buffers  8
/ t2 v6 c' X- ]  Filter Paper for Blotting  8 & P/ P+ h  S! ~/ |
  PVDF and Nitrocellulose Membranes  9
4 {* k0 h, J2 o/ v  Thermo Scientifc Pierce Protein Stains   9  5 I" c" q( R1 W
   for Membranes   
' X4 k  M. I& Z3 ~0 R- I. i; L) ~  Antibody Extender Solution NC  10 / }- p8 k2 P& p+ O( `
  Western Blot Signal Enhancer  11
/ f7 W) M/ o( }6 l3 A9 x% mStep 3 – Blocking
. |( B- q7 s' i( P" l/ {: O% m8 N2 ^  Introduction  12 5 l. E% U! A6 T3 v1 n
  Blocking of Nonspecifc Binding Sites on  12 $ u+ ^- j8 m( B( H# ~
   Transfer Membranes   
* M  x; d! k( V  Blocking Buffer Optimization  12-13 / z  q& S  S6 H# Z# E# r* m
  Blocking Buffers  13-16
$ N+ u# D. H- P" D% GStep 4 – Formulate Wash Buffers 3 s1 [2 ?6 T* B7 I$ ~" c  K
  Washing the Membrane  17 0 \/ B3 @. s& Z5 W! p* H' f, a6 `
  Wash Buffers  17& m6 \5 ]" V% o1 _, {0 u9 W$ Z
Step 5 – Detection Reagents / X2 c- Z% K! k; Q1 B" s& t5 F
  Validated Primary Antibodies  18-19 2 K0 q) Y4 X8 I5 {. a5 H5 x
  Affnity-purifed Antibodies  20
: n( X' J- s1 u9 s; [; ?  Stabilized HRP Conjugates  21
# @3 z, }# n% z5 v  Thermo Scientifc DyLight   22 # l& R/ R5 r: H: G5 d
   Fluor Conjugates   ; i# A( t) V9 {* i- ]+ r
  Conjugate Stabilizer Solutions  22
( X6 @- ?- u: w4 F2 u  Thermo Scientifc DyLight-labeled Highly  23
5 C: m, H2 v( _   Cross-Adsorbed Secondary Antibodies + S* Y; Q0 X9 _2 y/ N
  Antibody Storage and Stabilizer Solutions  24   K1 y& A8 Q) P: \1 y0 K
  Secondary Antibody Ordering Table  25-26  
6 u$ e: c' C/ Z% g% Q5 w  Thermo Scientifc Clean-Blot IP  28-29 % R( K% C% j7 i4 z" z/ i
   Detection Reagents   
7 F1 ^5 H& {0 n) A! n; o  Protein A, G, A/G and L Conjugates  30
5 k) |$ b6 @9 q8 Y  q- l  Thermo Scientifc NeutrAvidin, Streptavidin   31-33
& n% {4 R: g+ J   and Avidin Conjugates
. b5 b; p$ u+ |1 AStep 6 – Enzyme Substrates , [, L1 U3 M/ h
Chromogenic Substrates  34-35 ! l1 }& u! k0 N8 _- v( X+ [" o
Thermo Scientifc Chemiluminescent Substrates  36-43 2 ?" c% Y( T! ~+ o$ {- ]
  Pierce ECL Substrate  37 ! ~' J" t4 }$ o
  SuperSignal Chemiluminescent   38-42 5 ^- _9 l- y. Z+ k
   Substrates and Kits   
+ D4 Z6 ^: q  \# q  Lumi-Phos™3 Y) `- \" i. Z7 `% [7 \2 g
Chemiluminescent Substrate  43 ! g; s$ O- X- Z/ N8 R0 H: A' _2 ~
  Quick Reference Substrate Guide  43 4 B& L: `6 B1 P1 D
Fast Western Blotting Kit  44 5 F' q3 U( w4 c! N
Specialized Western Blotting Kits  45-48
1 A5 v2 t0 ?: F0 v! K" l  Thermo SuperSignal West Pico HisProbe Kit  45
8 D. Q& A2 U/ F  Pierce O-GLcNAc Western Blotting Detection Kit  46 , k$ J4 v* \; v
  Thermo Scientifc DyLight 549/649  46-47  
4 T  B0 }3 N7 b0 c* u9 A  M   Western Blotting Kit   " N- a6 l  V! u9 |+ S
  Thermo Scientifc DyLight 680/800 Near Infrared  47
" F& Q. X7 T) @' k6 e+ G, z   Western Blotting Kit   
4 X3 p7 w% P" ~# \  Thermo Scientifc Active GTPase Pull-Down  48 5 N! C) j9 k: X2 ^5 W- x) d4 w. O
   and Detection Kits   $ z$ A/ s5 f7 Q0 r; ^& `
Far-Western Blotting  49 0 S2 c2 ]5 d3 Y2 t0 x! i
In-Gel Western Detection  50-51 6 Q6 ^3 J  [* b6 x6 R
  Thermo Scientifc Pierce In-Gel   51
' @7 j$ n, ~. e6 q& I   Chemiluminescent Detection Kits
3 y2 b7 ]* O% P) L% A# _/ pStep 7 – Film 6 N$ Y, |$ k* M0 e
  Thermo Scientifc CL-XPosure Film  52
  E6 b  `. E* i7 c, O: o3 {Step 8 – Stripping Buffer 8 c2 G! Q" E: w0 x3 c
Optimizing the Signal-to-Noise Ratio  53-58 ' {$ R# O* q$ q$ [! Q  r' I
  Protocol for Stripping an Immunoblot  54
; G% z+ C$ r( |  Thermo Scientifc Restore  55 3 p  o- L: s* W+ H& S* m1 q" {5 S
   Western Blot Stripping Buffers $ U( ?" i5 ~3 R( J3 B
  Thermo Scientifc Restore PLUS  56
) ]! u: ~: y4 w  R1 J7 {   Western Blot Stripping Buffers
+ b) C% B( M# X6 P" m3 K* t  Thermo Scientifc Pierce   57-58 # `9 F! a6 V9 b% E# k( L
   Background Eliminator9 N4 e3 A9 Z' D
Troubleshooting Guide  59-69
7 |* _0 J' {2 ?, y5 ~" N8 _  Blotting with Chemiluminescence  59
: h, N3 p4 ^: P2 I  Optimizing Antibody Concentration  60-63 , T1 H) [' ?$ Z+ ^
  Problem Guide  64-67
3 b" |( U( f, f  M  Full-Length Western Blotting Protocol Using   68-69 , ?1 Y4 n6 }$ _1 l
   Chemiluminescent Substrates , e. P' X1 V6 V4 W
Recommended Reading  70" ^3 h: M1 L" h5 T, g+ ~0 R
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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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白衣小彩

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sunyucheng

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daviddow

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mominglily

谢谢

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