Western Blotting, a method for the identification of a specific protein, is based on a three-step approach: size resolution by gel electrophoresis, transfer of separated proteins to a membrane, and specific identification by labeled antibodies.

1.	Antibody is of low affinity or has lost activity during storage. For low affinity antibodies eliminate detergents from buffers. Increase the concentration of the primary antibody and the incubation time. Avoid repeated freeze-thaw cycles for antibody.
2.	Epitope may be conformation-dependent. Try with native and denatured protein samples or different antibody.
3.	Electroblotting was inefficient. Stain the gel or the nitrocellulose membrane after transfer. Increase transfer times and voltages. Make sure that pH and amount of SDS is appropriate. Switch to PVDF membranes.
4.	Inappropriate blocking buffer may have been used. Switch to a blocking buffer based on another protein (gelatin, casein, egg albumin, etc.).

This is the most common problem with Western blots.

1.	Concentration of detection reagents (secondary antibody, enzyme-conjugate) was too high. The high sensitivity of InnoGenex chemiluminescent substrates requires greater dilution of detection reagents than normally used for other chemiluminescent substrates. As a rule of thumb antibodies should be tenfold more dilute.
2.	Blocking was inadequate. Increase the concentration of the blocking reagent, the volume and/or the blocking time. The blocking reagent may contain enzyme activity or cross-reacting IgG or biotin-like activity. Switch to another blocking reagent. Make sure that the blocking reagent contains Tween-20 or Triton X-100 (0.05 to 0.1%).
3.	Washing was inadequate. Use sufficient volumes of washing buffer. At least four to six changes of wash solution are recommended. Transfer of membrane to a clean container for every wash is highly recommended.
4.	Target concentration was too high. To reduce signal intensity, wash membrane briefly in distilled water and incubate in substrate diluted 1:5 in PBS (for HRP) or 1x Activation Buffer (for Alk Phos).
5.	There was non-specific binding of detection reagents to the membrane. IgM and some other antibodies tend to be "sticky." Test on a nitrocellulose strip without any antigen. Secondary antibody may interact with other antigenic determinants. Dilution of the antibody may help.
6.	There is interfering enzyme activity in the sample. Inactivate enzyme activity by incubating membranes in 0.1M acetic acid for 20 min followed by washing in PBS.

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1994) Current Protocols in Molecular Biology. John Wiley & Sons.

Crowther, J.R. (1995) Methods in Molecular Biology, Vol 42 - ELISA: Theory and Practice. Humana Press, Totowa, New Jersey.

Davis, L.G., Kuehl, W.M., and Battey, J.F. (1994) Basic Methods in Molecular Biology, 2nd Edition. Appleton & Lange, Norwalk, Connecticut.

Price, D.C. (1996) Chemiluminescent substrates for detection of restriction fragment length polymorphism. Sci. Justice 36(4): 275-82.

Sambrook, J., Fritsch, E.M., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.