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Western Blot 步骤多，每步都可能出问题，所以，每步都要小心，多练习吧！祝好！* l5 n* [: V7 n3 f
% u/ f0 z& w: eTROUBLESHOOTING TIPS - WB
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U, m& u3 U4 J$ U' }No signal
6 m! x1 o1 _! p. |" NThe primary antibody and the secondary antibody are not compatible.
: h6 p D: i. h( r. K& n6 c) ]7 jUse secondary antibody that was raised against the species in which the primary was raised (e.g primary is
# f% x" Z4 r; c; M4 qraised in rabbit, use anti-rabbit secondary).2 Q2 U/ `. T/ u7 O0 D
. V( V1 e* S6 H Z0 JNot enough primary or secondary antibody is bound to the protein of interest.$ Y1 y" a3 Z) _# d* K( e' Z5 i
Use more concentrated antibody. Incubate longer (e.g. overnight) at 4ºC.8 k9 v6 R! r: E% V, _3 Y$ H7 E2 f. ]
, S3 q& ]; j$ U- h
Cross-reaction between blocking agent and primary or secondary antibody.9 |) ]' s/ {) J( U
Use a mild detergent such as Tween20 or switch blocking reagent (i.e. commonly used blocking reagents! z5 x: {/ W2 {, o% }
are milk, BSA, serum or gelatin).
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$ u' Y5 h q' @$ o) D, \% [The primary antibody does not recognize the protein in the species being tested./ J d' g8 {6 y/ ]
Check the datasheet or perform a ClustalW alignment to ensure your antibody should react with the target
+ Y4 L5 o; a2 gprotein; Run the recommended positive control.! [8 ]6 f6 y( {) o# B; `( L
* u8 e' [9 |3 H2 Z9 k! WInsufficient antigen.
, d0 w+ e) y% b5 x# Y2 d! ^) U* o1 GLoad at least 20-30 ug protein per lane; Use protease inhibitors; Run the recommended positive control.1 L9 B. D) K4 J; d
) S a6 K% n6 k% W
The protein of interest is not abundantly present in the tissue.
9 t2 l# t% h) RUse an enrichment step to maximize the signal (e.g. prepare nuclear lysates for a nuclear protein, etc.).; Q' W5 F; ~6 w
* Z- q% F3 ]: R/ e& O/ s4 t/ \0 O
Poor transfer of protein to membrane.' O1 y) C. ^5 j3 R. ^# @8 I
Check the transfer with a reversible stain such as Ponceau S; check that the transfer was not performed the' k) i& b8 n; c' c% d
wrong way; if using PVDF membrane make sure you pre-soak the membrane in MeOH then in transfer buffer.
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8 V9 f! I: A! {3 I5 o# e" x6 {Excessive washing of the membrane.
* Y7 a. ~- `$ y. Z' |Do not over wash the membrane.
# }# v- _" a+ K. h6 H$ MToo much blocking does not allow you to visualize your protein of interest.5 V, Q( r5 n& E
Instead of using 5% milk in the antibody buffers try removing the milk or using 0.5%; Switch blocking/ T, x# ]4 R' X1 a! W/ s P7 h
reagents or block for less time.3 \& z: l5 k; P" b! g0 m. b
( y: G8 T- |% p* t" s
Over-use of the primary antibody.
$ O8 a9 q* y1 E6 f( _! M8 O. tUse fresh antibody as the effective concentration is lowered upon each re-use.' z9 M" y- F" i% K& p( B, p0 B
. d; l8 |9 ~( OSecondary antibody inhibited by sodium azide.# t9 b3 [" F& b( n6 ^
Do not use sodium azide together with HRP-conjugated antibodies.; X ^( b8 |& Z6 ~
1 ]$ a: s; Q' X6 f
Detection kit is old and substrate is inactive.
+ Z' ^8 Z Z5 Q- r6 Y; bUse fresh substrate.
# t+ k) p; G: J9 G* G7 h! ]; W% g" ?
9 S; |+ K. B/ h, E+ p% x: s CHigh background
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Blocking of non-specific binding might be absent or insufficient.6 G/ N+ {/ q% m- i; F& ^% w
Increase the blocking incubation period and consider changing blocking agent. Abcam recommends 5%3 L& [$ b7 B4 l1 D6 p, B5 _; |5 ^
non-fat dry milk, 3% BSA, or normal serum for 30 min. These can be included in the antibody buffers as well.
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The primary antibody concentration may be too high.# x' I+ O7 G+ S& P, q7 n. E
Titrate the antibody to the optimal concentration, incubate for longer but in more dilute antibody (a slow but
. [/ W: f# D9 i' s) T7 ptargeted binding is best).
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7 n: m2 u( n& x1 KIncubation temperature may be too high.6 Y' I* L2 e+ L
Incubate blot at 4°C.
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! D. I9 [" H' q4 m# u4 e; V) uThe secondary antibody may be binding non-specifically or reacting with the blocking reagent.6 Z* e3 g2 R2 O8 C7 w& u' X
Run a secondary control without primary antibody.& C! N g- H% y) d! J- R
3 p5 z: W! M6 o M
Cross-reaction between blocking agent and primary or secondary.
+ z' }4 @( H1 ~( y: s( Q# V1 r: T0 [Add a mild detergent such as Tween20 to the incubation and washing buffer.
6 O5 R# e+ E% i0 C+ G g+ I+ {5 A(phospho-specific protein) Milk contains casein which is a phosphoprotein; this is why it causes
5 w; [; }" ?. _6 Q' U: ?; fhigh background because the phospho-specific antibody detects the casein present in the milk.; a& ~ V# s: w# a8 k, V
Use BSA as a blocking reagent instead of milk.
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( x D( w' o ], X) Z9 zWashing of unbound antibodies may be insufficient.& t9 C$ P- k* y% }) k
Increase the number of washes.
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# j) v8 @. o( Q/ x2 OYour choice of membrane may give high background.
5 k* f' P" [& X0 k3 @$ ^! LNitrocellulose membrane is considered to give less background than PVDF.
7 ]/ z! ~+ J9 T' a$ d6 i
- }3 B! A2 K) mThe membrane has dried out.
R. X" ]; _- w* bCare should be taken to prevent the membrane from drying out during incubation.
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Multiple bands, b k! U* g6 A/ w
# }( |$ y1 g4 {* X1 ~
Cell lines that have been frequently passaged gradually accumulate differences in their protein1 J+ \% A/ j! ^$ B
expression profiles.& D2 b: A" B9 E9 d- w. f! ]
Go back to the original non-passaged cell line and run the current and original cell line samples in parallel.* ^+ p; [0 x5 w S/ y0 W
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The protein sample has multiple modified forms in vivo such as acetylation, methylation,
4 ^& F5 f% m& N! q4 d" ^! Z% Hmyristylation, phosphorylation, glycosylation etc.1 U% |; H9 }0 e6 v
Examine the literature and use an agent to dephosphorylate, de-glycosylate, etc. the protein to bring it to. \4 X# [9 R6 i( X! s
the correct size.
! E0 C) M% ]# s+ ]) J2 N) ?1 c; P- L5 |+ c8 P$ Z( u
The target in your protein sample has been digested (more likely if the bands are of lower molecular& x* E D% X7 L0 w% C
weight).
; Y' p7 e: W4 |2 ]7 ~Make sure that you incorporate sufficient protease inhibitors in your sample buffer.$ A& F! K* m& W$ e+ ~0 I+ ~' h' j
# Q1 l% O# ~. L" ?; DUnreported novel proteins or different splice variants that share similar epitopes and could possibly
" s5 U; W- y# i. V! s' ]8 m! P# Abe from the same protein family are being detected.
' A& P( H! V) }2 }& M' b' a- jCheck the literature for other reports and also perform a BLAST search; Use the cell line or tissue reported& q% B2 Y: g u! O. a8 N
on the datasheet.* s9 F, s! T5 ]4 ], p5 }) A
+ W8 J3 K; x2 `4 u! X" \Primary antibody concentration is too high - at high concentration multiple bands are often seen.5 }6 R" \; i' u. g1 O
Try decreasing the antibody concentration and/or the incubation period.8 f6 L- \, k3 S1 w/ y: S) k% a
, J# Y+ |/ U9 m0 W( X8 Y/ T5 SSecondary antibody concentration is too high - at high concentration secondaries will bind
9 ?. C% V# q0 E1 e6 |( Gnonspecifically.
" L7 e! c" Z$ d$ b9 Z4 i6 ~# vTry decreasing the concentration. Run a secondary antibody control (without the primary).
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1 l: n3 i. `; W' `The antibody has not been purified.
7 ], M- m! a& W4 X0 z3 y G/ V6 i# NTry to use affinity purified antibody. This will often remove non-specific bands.% X& t% E' i8 e! F2 r2 B% [
% u2 r7 {6 ]- m- d8 i4 Z) ZThe bands may be non-specific.
0 T' M# U9 Z$ e" O/ u4 Q8 {9 nWhere possible use blocking peptides to differentiate between specific and non-specific bands. Only0 C/ S( d: c* [: K+ q4 v- E
specific bands should be blocked (and thus disappear).. G+ d5 t0 w4 ?3 ^( e
F5 U6 }+ C! c# M3 ^7 DThe protein target may form multimers.1 X) Z6 d; d# ~% f; d5 U) k: \' K
Try boiling in SDS-Page for 10 minutes rather than 5 minutes to disrupt multimers. N' B) K2 N/ i: X! O
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Uneven white “spots”on the blot
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; |" }: }, q3 \/ B4 d; N- ?1 u6 x0 CAir bubbles were trapped against the membrane during transfer or the antibody is not evenly spread
) c% x2 [) F* f- ?on the membrane.
! y) U- l# K1 z) D0 g! _Make sure you remove bubbles when preparing the gel for transfer. Incubate antibodies under agitation.! ]5 _& q3 G4 y5 m D
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Black dots on the blot
1 {+ G- B7 t/ p; E( [: t3 l- O7 w$ D
The antibodies are binding to the blocking agent.
2 s6 i" }# {7 k+ o; B3 Q7 C, _3 FFilter the blocking agent.
B' L; P/ \) f
' ^0 E5 s9 ]/ X4 |" i# P+ QWhite bands on a black blot (negative of expected blot)
0 |. i; D! |. Z7 ]
) I) N0 w- T6 u+ PToo much primary and/or too much secondary antibody.; f6 C- D6 ?2 }% A M
Dilute the antibodies more.
4 s7 [; P/ t9 l+ Q$ {9 _% J6 F; f9 `. } N" r! y0 g. z) a( o
MW marker lane is black
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2 w+ [( q4 c% j6 m2 R; lThe antibody is reacting with the MW marker.
; d! q! c) b$ C2 n' L# n2 TAdd a blank lane between the MW marker and the first sample lane./ d4 q6 Z$ Z3 b" u* ^7 L
8 U- I) I" V( b1 SThe band of interest is very low/high on the blot
: @. e( S$ c7 z
( A: z- G5 h+ `/ C. B4 y) w0 WSeparation is not efficient.; w3 t6 E, z' }" g5 B+ r
Change the gel percentage: a higher percentage for small protein, lower percentage for large proteins.% f \ d1 M: R8 S3 t4 I @3 L. t
6 U% [4 J8 I% j- G6 i4 W
Smile effect of the bands
! o( h: T: }+ y$ C& X( Q! f
) t$ @+ @- | ~( j1. Migration was too fast.6 ^8 O* {$ f7 ^
2. Migration was too hot (changing the pH and altering the migration).+ E0 s. L7 Z! ^- v
Slow down the migration or run the gel in the cold room or on ice.( ^) Y6 ~, }% V) ^5 V
* K0 F* e% M6 c! bUneven band size in lanes probed for the same protein
, X: N: a% X) X
& _& N* C. A! L* { QGel has set too quickly while casting and the acrylamide percentage is not even along the lanes.1 R+ Z2 p3 Y5 D* Z' L' _
Review the recipe of the gel and the addition of TEMED to the gels, add a little 0.1% SDS in water to the
7 x% k2 C7 a/ h2 f z9 Xtop of the migrating gel while it sets to stop it from drying.
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7 [- W% v) J p4 R# w9 BUneven staining of the gel) v% N2 _3 ~0 X6 n z
2 [$ ]( h* I1 I: W% K# ]
1. Contamination from bacteria
9 G3 y+ w- R' l3 u0 F. w3 e" _2. Not enough antibody
. w5 O2 s7 |# R9 ~' j' Z0 o1. Keep antibodies at 4°C and use fresh buffers covers the gel.# C5 m6 b, W! ]2 l& p6 l1 i# r d
2. Make sure the membrane is covered with the antibody/ incubate under agitation. |
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发表于 2011-2-24 15:46
发表于 2011-2-24 16:22
