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

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8 ^& w  l- y8 tIntroduction  13 t8 x. ]+ C$ P9 U2 x$ p
Western Blotting Overview  2-3
, f. ~! \2 i4 W+ h" R* v& N: tStep 1 – SDS-PAGE / Z+ s, D+ L/ g! P0 b/ s) B
  Thermo Scientifc Precise Protein Gels  4 ' g' ^' M- A1 N( _9 g2 d7 q
  Molecular Weight Markers  5-65 Q" Y$ Y2 c: [, ~; t# s
Step 2 – Electro-Transfer ' B' K7 B$ l. n; v! {0 K( U
  Thermo Scientifc Pierce Fast Transfer System  7 5 I! s$ G, Z7 p: y# N
  Transfer Buffers  8
9 }% k& N! k& q  Y- Y  Filter Paper for Blotting  8
4 R0 h+ b8 M$ h% g6 K9 ^: ]: ?  PVDF and Nitrocellulose Membranes  9 ) z# H7 \& V% r
  Thermo Scientifc Pierce Protein Stains   9  7 e, k& J9 V# ~
   for Membranes   * ^- b, V) Z! M. X* e! R& Y
  Antibody Extender Solution NC  10
$ s( C8 V0 {, x; `/ z: v  Western Blot Signal Enhancer  11
3 X. z5 g) c( lStep 3 – Blocking
8 s( w6 M6 ]8 h, R  z5 _  Introduction  12 ' a% C* a- ]/ n4 }" C
  Blocking of Nonspecifc Binding Sites on  12
7 A% p3 B' ?9 J, M0 x   Transfer Membranes   9 {, o# F. [, E( C' \
  Blocking Buffer Optimization  12-13
; ?! g* u. S0 `' [( V9 j  Blocking Buffers  13-167 `: c# F+ w2 M" e% s# k) A/ F6 A
Step 4 – Formulate Wash Buffers 7 j6 D7 S) Y; A: }- T
  Washing the Membrane  17
  a3 `6 f' i9 Z/ T6 N( b  Wash Buffers  17
; Y4 i, {5 X+ r' YStep 5 – Detection Reagents
9 M6 {8 S, W% ^# S  Validated Primary Antibodies  18-19
$ c5 ~5 E* x$ c" m" J  Affnity-purifed Antibodies  20 2 E% h9 A5 ~6 K+ j0 Z0 n0 t
  Stabilized HRP Conjugates  21 4 c1 C& u1 _& N+ O1 C3 A& I
  Thermo Scientifc DyLight   22 0 `  B6 e8 z, c) l$ Y* ]
   Fluor Conjugates   
6 i! J/ M5 }) m6 p  Conjugate Stabilizer Solutions  22 $ f& I0 M* e! h5 h, x
  Thermo Scientifc DyLight-labeled Highly  23
' Z5 ^6 w5 O7 S. C6 ?4 j   Cross-Adsorbed Secondary Antibodies . Q9 w- A, {/ C' T
  Antibody Storage and Stabilizer Solutions  24 ) @* D$ H7 \% x" @9 b! r# O
  Secondary Antibody Ordering Table  25-26  / P& y. \8 x1 y
  Thermo Scientifc Clean-Blot IP  28-29 * f; G6 [, M4 p! i% [
   Detection Reagents   1 ]! s- q# R$ k' n
  Protein A, G, A/G and L Conjugates  30
. \! t+ F% s) D4 L  Thermo Scientifc NeutrAvidin, Streptavidin   31-33
+ o+ R- J, z7 A0 G" {& }* U5 `   and Avidin Conjugates( X) X1 k6 v. ]( `
Step 6 – Enzyme Substrates
) b1 E. t  |. L/ `& A( h* A* rChromogenic Substrates  34-35
: j4 [  h) M" l. IThermo Scientifc Chemiluminescent Substrates  36-43 ) t/ N6 `6 ~: z- r9 w$ k' L
  Pierce ECL Substrate  37
, ~  M0 t* M( ^  A" A+ N! z, n  SuperSignal Chemiluminescent   38-42 , |7 x# l1 b% n! e1 g0 o. B$ ?. t
   Substrates and Kits   " v5 M+ t( n% _3 g
  Lumi-Phos™
% e# T) q# G( l) H Chemiluminescent Substrate  43
5 u$ \+ V5 c# I" a; ?0 K  Quick Reference Substrate Guide  43
& @; s' B: f, }$ oFast Western Blotting Kit  44 0 U& G0 B5 J' ?1 Q/ S+ l" {1 t
Specialized Western Blotting Kits  45-48
6 w% b; S. w3 t# |  Thermo SuperSignal West Pico HisProbe Kit  45 , R2 k: O5 `5 c. }( \
  Pierce O-GLcNAc Western Blotting Detection Kit  46
4 z% ^4 _3 I& c" j" v/ _  Thermo Scientifc DyLight 549/649  46-47  
6 v, h, G0 ^2 Q9 |( U5 n   Western Blotting Kit   
! s( ~9 n+ E. e$ U$ U% o  Thermo Scientifc DyLight 680/800 Near Infrared  47
& ?1 X& G1 j0 Z   Western Blotting Kit   
+ H8 H) W, X$ x# j' c* v1 v  Thermo Scientifc Active GTPase Pull-Down  48
1 A% @/ J9 u( `- N  ~/ d# k   and Detection Kits   . e- d$ J* u5 i( _: A1 {+ r2 g& m3 ?
Far-Western Blotting  49 3 |6 b6 Q! J/ u9 a' r% s" g! Q
In-Gel Western Detection  50-51
0 a1 q, a5 b, @# V  Thermo Scientifc Pierce In-Gel   51
4 H/ V6 k7 W; P2 Q8 J4 g% n   Chemiluminescent Detection Kits3 Z& W. L! M/ J+ K- X
Step 7 – Film / m0 U7 n2 |7 b3 S* u, W; h1 S
  Thermo Scientifc CL-XPosure Film  52
8 J6 ^+ t5 f- [: jStep 8 – Stripping Buffer 2 S# \) h% C, h
Optimizing the Signal-to-Noise Ratio  53-58
& m# e2 D6 C3 ~! v" l5 d  Protocol for Stripping an Immunoblot  54
& x, j, Z: H  I! U, `3 d# V. |  Thermo Scientifc Restore  55 / t. q2 _: i) i# U7 G
   Western Blot Stripping Buffers
# v' h/ K+ @; k4 Z7 Y  Thermo Scientifc Restore PLUS  56
6 W6 a) M+ H5 C; x( A   Western Blot Stripping Buffers + F1 j* @2 Y% ~6 {
  Thermo Scientifc Pierce   57-58 + Q+ y, X1 Z/ P) Q% M
   Background Eliminator
  ?4 J3 _$ G4 O) `* V/ H1 w! kTroubleshooting Guide  59-69
% |# g$ r( ?4 \  B1 ?! C. z  Blotting with Chemiluminescence  59
+ [+ v' b( s7 Y  o  Optimizing Antibody Concentration  60-63   {4 H9 X" s! f2 @$ `
  Problem Guide  64-67 % X2 s0 m0 Y/ M# r
  Full-Length Western Blotting Protocol Using   68-69 ; \1 T0 k3 r/ d: N
   Chemiluminescent Substrates ! E9 z! j- d/ a( x6 G/ b
Recommended Reading  70" g9 J: G) _4 p: g
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