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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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, E9 y k4 b% KIntroduction 1& g" F4 N/ u1 M* c: J
Western Blotting Overview 2-3# N. v0 m' { \, U+ y& f
Step 1 – SDS-PAGE * }; m( t; g# f. Q2 a/ t5 j0 q7 e9 l
Thermo Scientifc Precise Protein Gels 4 : {' N2 B5 g8 E! ^' i$ f4 B- o) P
Molecular Weight Markers 5-6
: c- p, k! z# A* x7 V! g: dStep 2 – Electro-Transfer
6 ? [' v+ F# Z3 x- {$ p Thermo Scientifc Pierce Fast Transfer System 7 $ J( ?; L$ W$ l1 v7 ^, \) }
Transfer Buffers 8
4 w. Q9 L j3 C7 u V8 c Filter Paper for Blotting 8
3 P, \# o3 g4 P4 @1 v# l PVDF and Nitrocellulose Membranes 9 6 v5 o+ ]! y3 M1 B5 l
Thermo Scientifc Pierce Protein Stains 9 4 Z0 g- j; R. V/ _" y* j; P1 d+ o/ o
for Membranes / U0 y% q/ o. T0 N( P$ u8 ~
Antibody Extender Solution NC 10
: G i f5 A& e9 [" F Western Blot Signal Enhancer 11
' d# ]+ ?& ]" H) f( @$ g4 EStep 3 – Blocking
( s$ H" f3 B' I# \/ L2 z0 @1 V+ Z Introduction 12
4 ?5 }' J. B* R9 I! y4 J- [ Blocking of Nonspecifc Binding Sites on 12
4 q! D N) J7 l |4 y Transfer Membranes + ?3 W* U% D; b' m4 ^! ^" T- f
Blocking Buffer Optimization 12-13 , m# J* _4 W8 h* N
Blocking Buffers 13-16$ M- F: b( B% F
Step 4 – Formulate Wash Buffers
7 Y" I3 h# h# E7 w Washing the Membrane 17 , C. e1 q: x R
Wash Buffers 17
* b* z, d' I* O, _- RStep 5 – Detection Reagents
' t/ x. _- j" i9 {% T1 n Validated Primary Antibodies 18-19
! {8 n$ B( W9 M% s1 \: k Affnity-purifed Antibodies 20
6 b4 |5 @5 L0 d: v: U: _4 Q Stabilized HRP Conjugates 21 , s5 ^; U. I* R# o3 h- l) \8 F
Thermo Scientifc DyLight 22
$ `4 ]# V$ f& ?; C/ [ Fluor Conjugates 1 V! r% N4 _( R' _; C
Conjugate Stabilizer Solutions 22 , B0 ?+ m: O$ C p {
Thermo Scientifc DyLight-labeled Highly 23
! \5 t0 Y( H+ t: `6 o Cross-Adsorbed Secondary Antibodies
: G. ^% \: w- z" ^. J/ H; W$ b Antibody Storage and Stabilizer Solutions 24 0 k) ?+ T' r& w% a5 ~
Secondary Antibody Ordering Table 25-26 # P/ ^7 _; X* r/ J
Thermo Scientifc Clean-Blot IP 28-29
, F- @# t% a' R2 K) v" z Detection Reagents
5 E1 N4 p- D) `4 y" [$ ?0 G0 V Protein A, G, A/G and L Conjugates 30 6 L, i+ O0 E9 K/ _2 o$ v
Thermo Scientifc NeutrAvidin, Streptavidin 31-33
3 A8 f' z1 z! p$ X. ] and Avidin Conjugates& J* ]% o; a; c' @' u, }* h, b
Step 6 – Enzyme Substrates
& Y+ s3 P- v2 ^7 Q! YChromogenic Substrates 34-35 * e K( R: F$ v: n
Thermo Scientifc Chemiluminescent Substrates 36-43 9 o4 @) a$ N% L* c$ ^
Pierce ECL Substrate 37
' i- [. O' X, u& R SuperSignal Chemiluminescent 38-42
0 p0 p' [% d3 I, t, z7 U, g Substrates and Kits
2 e9 b8 W4 i, G, E% V Lumi-Phos™
4 Y; K' c3 Q0 q1 t4 F" A Chemiluminescent Substrate 43
6 P( Q( M, r$ N+ S Quick Reference Substrate Guide 43 : r- n' l8 j" e [5 }. Y* u
Fast Western Blotting Kit 44 , G" R# Z, r& ~/ Y
Specialized Western Blotting Kits 45-48 % T1 o2 l; V- R7 j9 A
Thermo SuperSignal West Pico HisProbe Kit 45
7 p- \7 S$ P' ^( y3 M) ]5 w! z Pierce O-GLcNAc Western Blotting Detection Kit 46 : C N5 m8 W3 }* k
Thermo Scientifc DyLight 549/649 46-47
0 _. ^% l. Y2 Z. G5 T Western Blotting Kit
+ t- O u' v# S+ Q/ R- ]: Q Thermo Scientifc DyLight 680/800 Near Infrared 47 * ]; c* Z5 r9 [5 r. }+ i: c
Western Blotting Kit
) D% k( @. N* @4 y6 C, A$ Q2 [ Thermo Scientifc Active GTPase Pull-Down 48
/ Q9 J/ f: c9 A# F1 o% r and Detection Kits
8 L! b, |" \' `3 A+ `Far-Western Blotting 49 6 I6 j8 M. J U3 v
In-Gel Western Detection 50-51 & e$ N0 z* u( R( t8 Z
Thermo Scientifc Pierce In-Gel 51
) w. {" L6 X7 m0 i4 J" {" o/ V+ S Chemiluminescent Detection Kits
0 s7 ?) p w4 R' _Step 7 – Film
N1 n1 B" m3 ?: ^2 O& L Thermo Scientifc CL-XPosure Film 527 S) y- ^+ T' X# t5 t
Step 8 – Stripping Buffer . J" l; ^; ?4 G5 O) Y) u
Optimizing the Signal-to-Noise Ratio 53-58
0 G/ b5 L5 J! s+ E Protocol for Stripping an Immunoblot 54
7 ]: {* ?8 ~: p( p. ~ Thermo Scientifc Restore 55
5 B1 B5 {( Q0 j% B Western Blot Stripping Buffers # V; Q6 {! I# c4 D
Thermo Scientifc Restore PLUS 56
. K$ m; ?5 d. D+ ]# O Western Blot Stripping Buffers , t& D+ A# w% |& X$ X/ u
Thermo Scientifc Pierce 57-58 - u* h1 l+ C- X+ y7 x
Background Eliminator
; e" n, D8 V% ]6 B, X* B+ t% ?Troubleshooting Guide 59-69 0 ^# V8 d* |+ M0 _& }
Blotting with Chemiluminescence 59 1 n. d u4 d* _0 V) `0 P
Optimizing Antibody Concentration 60-63 9 `4 X" A+ F3 q
Problem Guide 64-67
9 l N% u2 Z# Y, {' o' ~& v Full-Length Western Blotting Protocol Using 68-69 0 B. u, A, n/ x$ }+ S
Chemiluminescent Substrates . c& @8 N8 \- P+ F0 }4 j
Recommended Reading 70
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发表于 2011-3-12 09:56
