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

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/ p7 c5 y) X, V  t$ U1 sIntroduction  1
: l1 p* _1 y" mWestern Blotting Overview  2-3
2 u( L/ D8 m' X9 ]/ LStep 1 – SDS-PAGE
# Y6 [: b9 G. ~4 s2 d  Thermo Scientifc Precise Protein Gels  4 , \( i" X9 Q; v0 m
  Molecular Weight Markers  5-6/ T; W  ]; L4 [$ c- U
Step 2 – Electro-Transfer , p' F+ A# E/ U+ N: }8 M
  Thermo Scientifc Pierce Fast Transfer System  7 5 O3 [2 N, {8 E! N& k9 b* n# c
  Transfer Buffers  8
* }- y% i  I9 y9 `  Filter Paper for Blotting  8
' L0 q; W+ |) ]6 x! E  PVDF and Nitrocellulose Membranes  9
1 r: q0 T7 {% X7 U$ b  Thermo Scientifc Pierce Protein Stains   9  , R% z( J4 s1 r% j  i
   for Membranes   ; s3 ^8 N$ E+ K
  Antibody Extender Solution NC  10 0 c5 k& S: ^6 ?# V; S: T  R& _
  Western Blot Signal Enhancer  11. M* r) v/ ]: F4 v5 x! p! |
Step 3 – Blocking
4 p% w) w% }4 O5 P* q) E  Introduction  12
, F+ E* C$ l/ |, O1 Z  Blocking of Nonspecifc Binding Sites on  12
- K! n6 t5 j, i: V+ z2 f   Transfer Membranes   
3 Z8 S+ {* {  `( C  Blocking Buffer Optimization  12-13
, ]) x* O! K$ y6 ^) z- D  Blocking Buffers  13-16
% R0 X2 R% ~. V. e# n2 oStep 4 – Formulate Wash Buffers
4 N) ?# o# \4 F  Washing the Membrane  17
( k4 l, `6 }" X/ W2 a4 r% E! U7 ^  Wash Buffers  173 z+ K0 R% ~9 S) N, u
Step 5 – Detection Reagents
7 g$ G- j$ v0 I# c1 S! }! ?8 u  Validated Primary Antibodies  18-19
; E9 I+ H, E4 M) x% D: t  Affnity-purifed Antibodies  20
+ \% M6 q  F" N  Stabilized HRP Conjugates  21
9 U. E8 y2 i/ t, U! n% x0 \) `" e  Thermo Scientifc DyLight   22
- b; r, g4 X' @" \6 J   Fluor Conjugates   
3 N8 M# S& p) P0 R+ J6 @  Conjugate Stabilizer Solutions  22 * t, k: E1 M; C% D' X
  Thermo Scientifc DyLight-labeled Highly  23
5 T) {# S3 r" d+ q3 a' D$ S1 l   Cross-Adsorbed Secondary Antibodies ' D$ Y0 t7 u0 d6 o5 D: V! r
  Antibody Storage and Stabilizer Solutions  24
/ T" Q6 ^# U7 v3 R6 K% E, E2 w  Secondary Antibody Ordering Table  25-26  
9 g7 f/ g+ c. ^( Y3 L! Z. \- X% s  Thermo Scientifc Clean-Blot IP  28-29 3 z5 ~4 @7 K+ K4 F& n
   Detection Reagents   : T- I3 n7 X+ f  d
  Protein A, G, A/G and L Conjugates  30 ; \- i! W1 b) P2 D
  Thermo Scientifc NeutrAvidin, Streptavidin   31-33
" b  Q; S/ i1 F   and Avidin Conjugates5 {' u" _) y; t/ G: Y
Step 6 – Enzyme Substrates # D+ O# \) h9 }3 `4 {
Chromogenic Substrates  34-35 1 y% U' x2 l0 i7 P8 m  Y0 i% C5 V" R
Thermo Scientifc Chemiluminescent Substrates  36-43 ! n8 e$ N0 |! j. o
  Pierce ECL Substrate  37
% Q; C8 Y- k9 e, x% B* n  SuperSignal Chemiluminescent   38-42
& y1 r9 y$ B) f- V, n   Substrates and Kits   / Y. J( A1 `: Z3 {8 Q3 l  s3 w
  Lumi-Phos™
; ^. M# k' R. q( G$ K9 ^; M4 j Chemiluminescent Substrate  43
; m( _: _" G5 a1 Y7 D7 U( ?  Quick Reference Substrate Guide  43 ' q$ I4 [8 f0 w9 [* f
Fast Western Blotting Kit  44 + Y# {, M; t( X' H; D1 R3 |* o' m5 @
Specialized Western Blotting Kits  45-48
, b0 H1 J2 R& ]* g0 O  Thermo SuperSignal West Pico HisProbe Kit  45 9 M1 L3 a1 O; k2 a7 ]- s
  Pierce O-GLcNAc Western Blotting Detection Kit  46
, d! U7 E9 C7 u% I+ o6 C; O2 M2 Z  Thermo Scientifc DyLight 549/649  46-47  : S" S/ E$ I! W
   Western Blotting Kit   , M5 P4 ~- P$ ?
  Thermo Scientifc DyLight 680/800 Near Infrared  47
( [: ~$ j! P3 J   Western Blotting Kit   2 b) h9 ]; h; s/ @5 J
  Thermo Scientifc Active GTPase Pull-Down  48
! y. H9 Y% \' f) Y( g6 {  D! c$ G   and Detection Kits   % B7 b( U, [+ |6 a4 R2 w7 M
Far-Western Blotting  49 6 Y, G  |( d" N$ u( o9 P- P5 e5 a0 v
In-Gel Western Detection  50-51 - J9 Z4 ], J% X) g, K0 Z
  Thermo Scientifc Pierce In-Gel   51
6 I! J3 V; I6 M% U6 P+ j0 {1 {   Chemiluminescent Detection Kits
7 l* H: i& Y8 Q% ?Step 7 – Film 7 P3 X# F3 k- B- i4 z; ^$ ?7 c0 |
  Thermo Scientifc CL-XPosure Film  52+ K" v1 g3 C" ]0 I9 T6 v8 M
Step 8 – Stripping Buffer
( Z8 `# Q1 R3 c/ n5 _0 U" @/ {Optimizing the Signal-to-Noise Ratio  53-58 4 r1 F! y6 e1 m( o, x4 m
  Protocol for Stripping an Immunoblot  54
9 A' ~, o8 e4 W0 q9 t  Thermo Scientifc Restore  55
4 T/ |" J3 c* B% p- ?$ t   Western Blot Stripping Buffers
- b) h9 v5 d9 F) S  Thermo Scientifc Restore PLUS  56
- F8 x- b9 A, a6 o6 g   Western Blot Stripping Buffers 9 d7 P/ U- z8 @$ V' n4 c
  Thermo Scientifc Pierce   57-58 3 U" }' t8 F4 v4 c. ^* N
   Background Eliminator
! G- |: f' O: j8 Q" iTroubleshooting Guide  59-69
! W. c8 j6 ?' J3 B9 W0 Q8 d  Blotting with Chemiluminescence  59 6 ~  A( ~4 k1 k' D
  Optimizing Antibody Concentration  60-63
) m1 [5 y% k+ s+ J  Problem Guide  64-67
3 {4 V& b/ K; F- \' z6 \! o: w  Full-Length Western Blotting Protocol Using   68-69
0 Q: `; ]- }4 z) h: R3 \$ ?0 ^   Chemiluminescent Substrates
6 Q0 s/ f/ R* X7 G% K/ M) F0 k: q0 q6 MRecommended Reading  70
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  Q$ A; f# g) {* U[hide][/hide]

一枝独秀

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jiandong2001

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军医

谢谢分享！

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

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