Antibody Guarantee - Western Blotting
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High Background
When extra bands appear below that of the desired protein, the most likely explanation is that they originated from proteolytic breakdown of the desired protein. This can be prevented by treating the sample with protease inhibitors during tissue or cell sample preparation.
Sample buffers with insufficient SDS and/or reducing agent (DTT, 2-mercaptoethanol, BMS) may not fully dissociate, reduce or denature a protein. Hence, extra bands may appear above the desired protein on the gel. Boiling the sample in sample buffer immediately prior to loading can reduce these noncovalent interactions or disulfide linkages. Use of an irreversible reducing agent such as TCEP, instead of DTT or â-mercaptoethanol, may also be helpful.
Sometimes, the bands are specific; for example, an antibody made against a peptide coupled to BSA may recognize traces of BSA in the sample. Homologous proteins may also be recognized by the antibody. When using a polyclonal serum, some bands may be due to the presence of antibodies produced as a result of animal's exposure to similar antigens in its life. It is best to run a normal serum control to determine the specificity of any antibody.
For anti-phosphoserine, phosphotyrosine, and phosphothreonine use blocking agents other than milk, as it contains numerous phospho-proteins that lead to high background.
Weak/No Signal
In some cases the primary or the secondary antibody might have lost its activity during storage or by repeated freezing and thawing. ELISA, immunoprecipitation, or another appropriate immunochemical assay can be used to confirm the low antibody reactivity.
In other cases the antibody may have low affinity. Here one can increase the incubation period and the concentration of primary antibody.
Weak signal can also result from an inefficient transfer of antigen out of the gel and inefficient binding of antigen to the membrane. This is caused by insufficient contact due to the presence of air bubbles between the gel and the membrane or an excess of SDS in the gel or buffer.
Other
The sensitivity of western blotting is dependent, in part, on the efficiency of transfer from the polyacrylamide gel onto the nitrocellulose membrane. In general, thinner and lower percentage gels give greater transfer efficiency and thus higher western blotting sensitivity. For proteins <20 kDa, use a 0.2 µm rather than a 0.45 mm nitrocellulose filter.
Nonfat powdered milk is an excellent blocking agent, but other proteins, such as casein, gelatin (Cat. No. 345808), BSA, and ovalbumin, may also be used. For anti-phosphoserine, phosphotyrosine, and phosphothreonine it is recommended to use blocking agents other than milk. Milk contains numerous phospho-proteins that lead to high background.
Membranes should be rocked during all washing and incubation steps
Any wash step may be extended to overnight at 4°C
Blocking may be carried out overnight at 4°C
The concentrations of primary and secondary antibodies should be titrated to determine the optimal concentration.
If the substrate solution develops precipitates during storage at +4°C, warm it to room temperature and mix; a sonicating water bath may also be used; a small amount of precipitate in these solutions will not harm the performance of the product.
Improved performance may be obtained using subcellular fractions for Western blotting, i.e. nuclei for nuclear proteins or membranes for membrane receptors; concentration of antigens by immunoaffinity chromatography or immunoprecipitation aids in obtaining more intense bands on Western blots.
If the problem you are observing is not accurately described or remedied by any of the above suggestions please let us know using this form
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