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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！/ S" @0 }. O) v( p Y1 X
0 x& a6 F. o8 y: j8 jTROUBLESHOOTING TIPS - WB+ P1 s$ }! v- C3 n4 x" R
/ v0 r6 n7 W; e/ n! U* X7 kNo signal
4 o6 p9 `/ I+ d5 ~; CThe primary antibody and the secondary antibody are not compatible.4 H: i% I9 ]# p8 f6 F8 N0 h/ g$ u
Use secondary antibody that was raised against the species in which the primary was raised (e.g primary is
" Z3 v) Z1 x7 t, f: X; k: S, a2 wraised in rabbit, use anti-rabbit secondary)." ]7 k; x5 E( p: Q/ c
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Not enough primary or secondary antibody is bound to the protein of interest.2 ~# U: I! j6 p
Use more concentrated antibody. Incubate longer (e.g. overnight) at 4ºC.
' |) n) q r3 N" U% m# E) Q6 \5 s" q( V2 v" A* W/ r4 c3 R/ `: R
Cross-reaction between blocking agent and primary or secondary antibody.
' T' E r2 u6 jUse a mild detergent such as Tween20 or switch blocking reagent (i.e. commonly used blocking reagents
9 x6 V8 m# P8 D* ^are milk, BSA, serum or gelatin).+ ^6 S3 {+ {+ K1 [+ B* {
8 C* j9 ^/ y/ i9 O4 @$ qThe primary antibody does not recognize the protein in the species being tested.
0 Q* n4 O( M6 `1 G8 L9 [Check the datasheet or perform a ClustalW alignment to ensure your antibody should react with the target4 z% y% K1 d2 y* H6 h
protein; Run the recommended positive control.
1 {/ `: ~1 C9 s. o9 I7 j3 P" H+ k/ t- w0 A+ d, _! C
Insufficient antigen.
1 H6 U- `6 I+ S. h' V# ^Load at least 20-30 ug protein per lane; Use protease inhibitors; Run the recommended positive control.( V4 o- k3 E; n* s7 W1 Y r
0 p+ N7 V8 H$ Y' J6 i
The protein of interest is not abundantly present in the tissue.
2 s1 l: J% o0 j! k- KUse an enrichment step to maximize the signal (e.g. prepare nuclear lysates for a nuclear protein, etc.).
! f p6 m1 h( l
+ L% I7 [( s2 R6 uPoor transfer of protein to membrane.
# @, M+ C3 R# H1 i( B# m" k) `- CCheck the transfer with a reversible stain such as Ponceau S; check that the transfer was not performed the) ^, Z$ W% A" F4 d: O- A
wrong way; if using PVDF membrane make sure you pre-soak the membrane in MeOH then in transfer buffer.
1 \) H7 L' X; i" ^1 p( Y+ l
5 @& i5 E- s* V, O4 S; _! \* UExcessive washing of the membrane.
$ r1 k* O" _# y9 O& m7 Y+ K. q4 TDo not over wash the membrane.8 Z' L( W- y# {! _8 U$ X- t' z" P; y% a0 ` b
Too much blocking does not allow you to visualize your protein of interest.
) b1 ~, s* ?" {2 a2 ^, b% u6 pInstead of using 5% milk in the antibody buffers try removing the milk or using 0.5%; Switch blocking K( X) I. H9 m* e7 W
reagents or block for less time.
& k* m- i3 S2 m: u; t6 I4 x
" w& D m0 T- d' nOver-use of the primary antibody.8 [5 m2 W: T; S, a9 X) t8 J
Use fresh antibody as the effective concentration is lowered upon each re-use.
( A+ r! `( f" l& o
1 g4 U9 _6 S r5 ~/ ~ E, x! ASecondary antibody inhibited by sodium azide.9 P+ r4 [+ O+ A6 O
Do not use sodium azide together with HRP-conjugated antibodies.
4 m4 Y! `- n* K3 C: H7 C6 E
$ v# ~$ _" i/ u3 x; X5 I& T4 X xDetection kit is old and substrate is inactive.' f4 z6 |) y- Q: e5 }* K
Use fresh substrate.& P t4 h8 u Q. f0 A/ z
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High background
7 R5 l3 f/ q/ S; B9 S. Y' i. `& c9 U8 z9 [( @2 T* o
Blocking of non-specific binding might be absent or insufficient.% Y, w- a! i' B" O) o
Increase the blocking incubation period and consider changing blocking agent. Abcam recommends 5%
, ~ a: P: `" bnon-fat dry milk, 3% BSA, or normal serum for 30 min. These can be included in the antibody buffers as well.
q% b" [( V" n* D0 B5 A% d- r
. B2 U; P4 w& {8 b; j; f( ]; mThe primary antibody concentration may be too high.
8 W, ~( s( ~, d/ I$ JTitrate the antibody to the optimal concentration, incubate for longer but in more dilute antibody (a slow but
6 y' j4 k6 o$ }6 ~0 ytargeted binding is best).
9 H: T$ R% H1 Q8 [, S' \2 V1 l; c+ a* B% k8 w
Incubation temperature may be too high.
4 N# K. u) ?; D6 w# WIncubate blot at 4°C.
& m# Z6 {2 K6 N+ l# M
3 a% u; b- R; _' f$ W; EThe secondary antibody may be binding non-specifically or reacting with the blocking reagent.
. t7 z8 }0 G! ?1 Q% N4 u7 g0 n" {, XRun a secondary control without primary antibody.0 o! W" K, u: c, W( m# K
8 o: c9 O8 T( {$ f+ f+ MCross-reaction between blocking agent and primary or secondary.% t5 o( p; n h& U
Add a mild detergent such as Tween20 to the incubation and washing buffer.
9 L) u% E9 W, b5 `& t% z(phospho-specific protein) Milk contains casein which is a phosphoprotein; this is why it causes
$ ]; e; C8 O8 |# c- q2 Hhigh background because the phospho-specific antibody detects the casein present in the milk.
* M+ i* R3 c: J. o% n: f% `" P: uUse BSA as a blocking reagent instead of milk.8 i4 }) ]. m( z/ _# r7 e0 X* m
5 `! B! P' R+ @4 [! \/ }0 s1 LWashing of unbound antibodies may be insufficient.
, Y* u! L c; Y6 e! P% MIncrease the number of washes.
1 }3 s; J3 n- l- h+ U
7 \1 U# U3 S# V# d' J- MYour choice of membrane may give high background.
# n* z7 m: v5 `) N. gNitrocellulose membrane is considered to give less background than PVDF.' H! ]9 }) F- G+ A8 M1 e
- \& E* A8 B# lThe membrane has dried out.
/ j( s$ O* W7 b1 L5 P, I; dCare should be taken to prevent the membrane from drying out during incubation.
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Multiple bands: k0 j# b$ d+ G# w3 O/ ^
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Cell lines that have been frequently passaged gradually accumulate differences in their protein8 A# M5 o! D+ w# u% U4 F# R8 j
expression profiles.
" p6 |+ M% e; y& h! {% D2 r% jGo back to the original non-passaged cell line and run the current and original cell line samples in parallel.
! L7 j+ j# p" X3 R& M6 m; }
1 y& L2 \4 d% N2 N% }The protein sample has multiple modified forms in vivo such as acetylation, methylation,. G5 E; o. J$ T: S* A+ f1 ]5 f* O+ a
myristylation, phosphorylation, glycosylation etc.
4 _% t( a6 @* { n" q+ A5 aExamine the literature and use an agent to dephosphorylate, de-glycosylate, etc. the protein to bring it to4 Q1 v7 m% [& A7 X) w4 }( t
the correct size.. }2 l! Y" U1 M
" \8 M. T0 _4 C0 p0 W% z U t
The target in your protein sample has been digested (more likely if the bands are of lower molecular4 W: ^7 Q8 t4 ^# l
weight).
2 t9 q3 U. x+ OMake sure that you incorporate sufficient protease inhibitors in your sample buffer.9 J+ F$ a2 W, N
2 s/ k7 i% p, X# E& F0 ]Unreported novel proteins or different splice variants that share similar epitopes and could possibly8 L, A3 R- m: ^, G' F
be from the same protein family are being detected.4 J1 i1 K& {: F/ E9 }
Check the literature for other reports and also perform a BLAST search; Use the cell line or tissue reported. ^$ V# _. p* @( X
on the datasheet.5 }. ]* `; b! |( E. D% S% F7 |7 r
( q+ G' x6 B1 `7 Y. N* T% h! M' V
Primary antibody concentration is too high - at high concentration multiple bands are often seen.
. S9 P3 v8 s% }% R2 A7 n/ X. LTry decreasing the antibody concentration and/or the incubation period.
; h* f* J0 U6 F) j! n- j" ?0 {" {$ I8 J. C$ c3 Q" Z$ ]7 l) F
Secondary antibody concentration is too high - at high concentration secondaries will bind
; p& ^( N9 i1 c( Q" [/ O3 Hnonspecifically.5 ~, k0 Y2 B/ y" Y# `4 D
Try decreasing the concentration. Run a secondary antibody control (without the primary).; ]& `2 w6 \ z
) }% N1 ^ f1 |. C/ ^' I4 b6 |The antibody has not been purified.
+ ~6 g* q u/ kTry to use affinity purified antibody. This will often remove non-specific bands.
Q: q6 \3 b! ]
4 p/ X7 ]9 N, V2 ^9 d7 XThe bands may be non-specific.$ {) B% K/ ^: q, M. j
Where possible use blocking peptides to differentiate between specific and non-specific bands. Only" Q& e" [8 L) W1 t0 ]3 |) t. L5 s
specific bands should be blocked (and thus disappear).
y* q: u( q I- ^
: p6 o) S: F: Z9 OThe protein target may form multimers.& a2 n; q1 K2 ?; H
Try boiling in SDS-Page for 10 minutes rather than 5 minutes to disrupt multimers.
A6 j1 t+ c( P
- h6 H- {& C- c3 Q FUneven white “spots”on the blot
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Air bubbles were trapped against the membrane during transfer or the antibody is not evenly spread0 X& y s# L; o8 U7 e/ D
on the membrane.
" P' @# f$ ~! bMake sure you remove bubbles when preparing the gel for transfer. Incubate antibodies under agitation.( N+ M+ G+ g4 {5 b# T, a& r. Y
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Black dots on the blot. q; `2 v% e/ p# W/ Q
9 W* e( a, }: _* P$ e+ _The antibodies are binding to the blocking agent.
4 v1 b2 }- z# j, {& V! i6 |; vFilter the blocking agent.
: s% V- z v0 L/ v3 o9 Z: V4 ]* r
7 _' }8 n ~$ ~' IWhite bands on a black blot (negative of expected blot)# z- a1 a% B/ j5 F, d$ g1 H1 c
8 l6 t: c9 p6 s5 z8 }0 Z! v
Too much primary and/or too much secondary antibody.2 ?7 G, O. L$ o% r. w: V/ D
Dilute the antibodies more.
6 |+ o( X: j, w
0 x! E0 x7 C$ Y# TMW marker lane is black% @; t! ]- d' |. {9 T6 t( n: J
+ x6 U% k7 \% L2 c
The antibody is reacting with the MW marker.+ }* A8 e2 ?4 c/ I2 R- F& K
Add a blank lane between the MW marker and the first sample lane.8 c' }4 F6 c( i3 i2 R" C$ O! |
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The band of interest is very low/high on the blot3 E" ]% U6 P7 B5 \: L/ }% K8 Z
% z( L) P4 o: TSeparation is not efficient.
7 e, G1 |! R- z7 U+ \7 l' x! jChange the gel percentage: a higher percentage for small protein, lower percentage for large proteins.3 s+ X& n. e3 q4 o! ?- S. e1 G
. R0 _$ [' s4 r, J# O4 F$ tSmile effect of the bands! ~% ^ _2 V+ z# Y5 W
# }8 ~9 E& _& q8 y# N0 |- r
1. Migration was too fast.
0 b; ~+ h: T2 T! ~, T5 R4 B: U2. Migration was too hot (changing the pH and altering the migration).
! R7 y5 u# g" x: h rSlow down the migration or run the gel in the cold room or on ice.
, v* i I9 L3 o3 A* F2 {; k% E3 ?1 u
& q* L) U, q- V/ S' y2 I& JUneven band size in lanes probed for the same protein
+ J* v$ l( c# D, L( r' B) _" G* J( ^6 M& \& P* J% w% b
Gel has set too quickly while casting and the acrylamide percentage is not even along the lanes.
# A2 U+ _1 I, v0 UReview the recipe of the gel and the addition of TEMED to the gels, add a little 0.1% SDS in water to the9 g* [; @1 z5 i9 x7 T
top of the migrating gel while it sets to stop it from drying.
+ F* n4 L+ S7 S$ O! Y+ S, i5 m i$ i# E: ]! U' o
Uneven staining of the gel
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$ F2 A) ?( A$ f0 B; E1. Contamination from bacteria, z/ e1 B6 e* T; L) ?- g
2. Not enough antibody, w2 K& J1 {* u
1. Keep antibodies at 4°C and use fresh buffers covers the gel.4 L+ Z, U8 O' `/ a, Y
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
