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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！
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! g2 k v: S# A) ]! ?TROUBLESHOOTING TIPS - WB
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No signal
, C8 t' a9 ~% J4 G- [; C2 fThe primary antibody and the secondary antibody are not compatible.2 l6 g& d6 _7 q( `
Use secondary antibody that was raised against the species in which the primary was raised (e.g primary is8 i# q& R* _$ v9 j0 m
raised in rabbit, use anti-rabbit secondary)./ w, X9 J; c6 [4 q
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Not enough primary or secondary antibody is bound to the protein of interest.* A5 T& W; ?* z) H* h8 _9 m% j
Use more concentrated antibody. Incubate longer (e.g. overnight) at 4ºC.
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Cross-reaction between blocking agent and primary or secondary antibody., j1 u9 E) d; ^! y! A
Use a mild detergent such as Tween20 or switch blocking reagent (i.e. commonly used blocking reagents5 k. r$ @0 B: N0 p3 U9 T
are milk, BSA, serum or gelatin).6 z" t `0 P8 R, b- v
+ d4 L. U$ z4 Z( ~# f# J! hThe primary antibody does not recognize the protein in the species being tested.( M' c7 v- y: p' ~' ^# W
Check the datasheet or perform a ClustalW alignment to ensure your antibody should react with the target% C8 s }/ ^; L( w
protein; Run the recommended positive control.3 `% v" o3 Y N$ t
* R2 Q2 C, M9 ]$ F% ]* ~) A- kInsufficient antigen.3 D. y& Q o8 ^# D1 d
Load at least 20-30 ug protein per lane; Use protease inhibitors; Run the recommended positive control.
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The protein of interest is not abundantly present in the tissue., t$ g8 b# O. I7 `3 R# z+ U
Use an enrichment step to maximize the signal (e.g. prepare nuclear lysates for a nuclear protein, etc.).8 \+ Z# D# _, S1 v2 L
3 \. h" m+ t0 b% @! A4 G7 bPoor transfer of protein to membrane., f" M) E) h7 U1 j4 P& [6 Z
Check the transfer with a reversible stain such as Ponceau S; check that the transfer was not performed the. |) Z4 L3 C) ?8 B, r
wrong way; if using PVDF membrane make sure you pre-soak the membrane in MeOH then in transfer buffer.
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% U" ^8 Q6 t0 `! q6 \- EExcessive washing of the membrane.
/ B& N% [! j. v, t9 n4 }0 zDo not over wash the membrane.
9 t- Z+ Q8 |8 w. [ v- x% ~Too much blocking does not allow you to visualize your protein of interest.( f8 ?; E; W( E* `* x' d' X5 G
Instead of using 5% milk in the antibody buffers try removing the milk or using 0.5%; Switch blocking
/ Q0 t# a0 G) Q4 dreagents or block for less time.5 R" v* X; f' d, H7 t0 l h
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Over-use of the primary antibody., D7 C' ]+ x8 H* k# A
Use fresh antibody as the effective concentration is lowered upon each re-use.
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Secondary antibody inhibited by sodium azide.
3 C/ K0 r' e+ Z5 V. kDo not use sodium azide together with HRP-conjugated antibodies.
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Detection kit is old and substrate is inactive.% Z/ I& a% O( s; H3 D7 N
Use fresh substrate.
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" l5 `3 J6 _. zHigh background
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6 I0 y2 a. R+ r& a3 X+ KBlocking of non-specific binding might be absent or insufficient. {5 ^" M0 e5 B) Q/ X$ V
Increase the blocking incubation period and consider changing blocking agent. Abcam recommends 5%: `2 s& r0 O* O
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.
3 ]% b7 v- Q+ @0 b8 t7 O* O2 U( eTitrate the antibody to the optimal concentration, incubate for longer but in more dilute antibody (a slow but
0 s# n6 I. {( P8 d R3 q/ ], Vtargeted binding is best).0 M/ T2 N8 x. \. s" r
1 L4 V0 L- H% mIncubation temperature may be too high.
5 e; T+ Q$ U9 Z2 T, FIncubate blot at 4°C.0 X0 r7 f6 J; A: }8 v
! a* A4 x) G) P$ R7 B' S ^The secondary antibody may be binding non-specifically or reacting with the blocking reagent.
' l. V. Z+ s& w: @! gRun a secondary control without primary antibody.
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Cross-reaction between blocking agent and primary or secondary.
% C6 T% Z7 Z# [ Z6 eAdd a mild detergent such as Tween20 to the incubation and washing buffer.
% g6 Y, i7 p3 G- Y. u2 o(phospho-specific protein) Milk contains casein which is a phosphoprotein; this is why it causes
3 @% k p% ^0 e6 K" A$ ^$ A' q: vhigh background because the phospho-specific antibody detects the casein present in the milk.
8 c1 b. N; z' m0 m8 ?' [4 _; gUse BSA as a blocking reagent instead of milk.
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3 }/ B) ^8 N* I1 Z7 E3 _9 QWashing of unbound antibodies may be insufficient.
& n2 R0 F0 e) |" I8 J: AIncrease the number of washes.% \; e9 k+ W" \; M& ^
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Your choice of membrane may give high background.
7 E7 F2 X$ K, xNitrocellulose membrane is considered to give less background than PVDF.! |& B; \9 c/ ]/ D% O# _1 _
& k1 l6 O; G1 w7 i; b9 R- DThe membrane has dried out.9 l; x9 x* N# {1 E. k* M
Care should be taken to prevent the membrane from drying out during incubation.
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- t6 X, b. t" A% I5 m. B$ x- TMultiple bands, h+ t" j. |) ?3 m
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Cell lines that have been frequently passaged gradually accumulate differences in their protein- B. R- p8 c* G7 l0 C$ ~$ `
expression profiles.6 G' A8 A8 \+ u) P1 b6 I
Go back to the original non-passaged cell line and run the current and original cell line samples in parallel.
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The protein sample has multiple modified forms in vivo such as acetylation, methylation,
' b2 e, U1 n) H# \3 Y( `myristylation, phosphorylation, glycosylation etc.
' R" ]* {3 I% X: G6 uExamine the literature and use an agent to dephosphorylate, de-glycosylate, etc. the protein to bring it to
! g( ^6 R1 {" `6 v% s* ^the correct size.
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7 U( v) |7 A" K5 E* P' ^The target in your protein sample has been digested (more likely if the bands are of lower molecular
9 ]5 o3 b6 m! E' ]3 U) x( tweight).
% A! q, Z/ c! WMake sure that you incorporate sufficient protease inhibitors in your sample buffer.) U- _" A# Q* w$ ^0 E0 S$ U j
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Unreported novel proteins or different splice variants that share similar epitopes and could possibly
' x+ @2 e o/ \& Q; j8 W+ Sbe from the same protein family are being detected.
2 I( J( Y- P% j" E1 c/ RCheck the literature for other reports and also perform a BLAST search; Use the cell line or tissue reported1 L" t8 W% {% U( \3 T: y5 f
on the datasheet.
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9 M1 x" ?& w1 U \Primary antibody concentration is too high - at high concentration multiple bands are often seen.
* L( Z( Q. d" j/ E# STry decreasing the antibody concentration and/or the incubation period.
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Secondary antibody concentration is too high - at high concentration secondaries will bind
+ T- d4 h3 O7 _' f6 d. |% ynonspecifically.
" a; V9 J9 R( r7 L& b8 TTry decreasing the concentration. Run a secondary antibody control (without the primary).
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The antibody has not been purified.
& \- p2 o* q9 v. J7 h$ i1 C3 n. BTry to use affinity purified antibody. This will often remove non-specific bands.
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2 T# z1 w, ~# I- n1 H9 c& I4 iThe bands may be non-specific.
$ S" @4 o: s) K" }Where possible use blocking peptides to differentiate between specific and non-specific bands. Only
" I5 |$ b! N7 A$ u8 ~specific bands should be blocked (and thus disappear).* x2 ^; g, @6 C' c- M C2 V" ?2 z5 B
4 ?6 S/ N! n1 q x5 @The protein target may form multimers.
2 w8 g# }3 s) b! A4 d9 dTry boiling in SDS-Page for 10 minutes rather than 5 minutes to disrupt multimers.# M( ] r$ l# C4 ^" ?1 T
) i ^3 ]# C/ x* e2 e& zUneven white “spots”on the blot
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' I1 q2 E) p' L% j3 JAir bubbles were trapped against the membrane during transfer or the antibody is not evenly spread, ^$ M9 a$ K! G' Q. p: H! l
on the membrane.
4 C( w p6 @& M8 Q2 ~, zMake sure you remove bubbles when preparing the gel for transfer. Incubate antibodies under agitation.1 w8 ]! s9 [) O) Y8 U2 u. V, i5 q
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Black dots on the blot/ O$ `1 j" J' [! d2 p B/ h
: {/ C/ ?" i4 M+ Q9 NThe antibodies are binding to the blocking agent.& r- f" C' A/ Q2 h, j; f" l
Filter the blocking agent.
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White bands on a black blot (negative of expected blot)( L. D2 T' u0 `# a' ^0 c% Y
+ m) I* T, Z) p/ U% ~3 P, UToo much primary and/or too much secondary antibody.
/ A4 m6 _; u" n, U- p* G. YDilute the antibodies more.
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MW marker lane is black: [ U! V9 o3 ^
- V8 a$ o& |8 j5 xThe antibody is reacting with the MW marker.
) x$ n$ C' F0 ]: W# VAdd a blank lane between the MW marker and the first sample lane.
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) ]4 c$ q2 F" J8 r2 T* {: WThe band of interest is very low/high on the blot- q5 O! \+ p) S; [9 E
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Separation is not efficient.( s9 k+ x# ]& {" h4 O
Change the gel percentage: a higher percentage for small protein, lower percentage for large proteins.
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Smile effect of the bands
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1. Migration was too fast.
: c; K7 S1 a% {1 ]4 _. _* @# {6 \2. Migration was too hot (changing the pH and altering the migration).
6 @3 s4 o! P& M% G7 L! pSlow down the migration or run the gel in the cold room or on ice." m! a+ Y. Z4 {
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Uneven band size in lanes probed for the same protein
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% ~) d% d) {; F5 r* ^3 j/ W6 W vGel has set too quickly while casting and the acrylamide percentage is not even along the lanes.% ~1 x( D( @; j5 c
Review the recipe of the gel and the addition of TEMED to the gels, add a little 0.1% SDS in water to the- f: G- k- G* Z$ K7 `& R! ]5 u
top of the migrating gel while it sets to stop it from drying.
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Uneven staining of the gel
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: A( c! N: e7 e& k( w1. Contamination from bacteria: J" \- @+ G& W% ~3 G) ]
2. Not enough antibody
4 Q; ~/ Q) m6 X; `' T- P1. Keep antibodies at 4°C and use fresh buffers covers the gel." g) E3 e% x8 x- X* b
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
