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wdwd123(½ð±Ò+8,VIP+0):лл 4-25 19:54
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1Blocking of non-specific binding might be absent or insufficient.
Increase the blocking incubation period and consider changing blocking agent. Abcam recommends 5%
non-fat dry milk, 3% BSA, or normal serum for 30 min. These can be included in the antibody buffers as well.
2The primary antibody concentration may be too high.
Titrate the antibody to the optimal concentration, incubate for longer but in more dilute antibody (a slow but targeted binding is best).
3Incubation temperature may be too high.
Incubate blot at 4¡ãC.
4The secondary antibody may be binding non-specifically or reacting with the blocking reagent.
Run a secondary control without primary antibody.
5Cross-reaction between blocking agent and primary or secondary.
Add a mild detergent such as Tween20 to the incubation and washing buffer.
6(phospho-specific protein) Milk contains casein which is a phosphoprotein; this is why it causes high background because the phospho-specific antibody detects the casein present in the milk.
Use BSA as a blocking reagent instead of milk.
7Washing of unbound antibodies may be insufficient.
Increase the number of washes.
8Your choice of membrane may give high background.
Nitrocellulose membrane is considered to give less background than PVDF.
9The membrane has dried out.
Care should be taken to prevent the membrane from drying out during incubation.

[ Last edited by sutao1979 on 2009-4-26 at 18:54 ]
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wdwd123(½ð±Ò+8,VIP+0): 4-25 17:01
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