Western Immunoblotting Troubleshooting Guide
Problem: High Background
General background is high or nonspecific bands appear after a 1–30 second exposure of blot to film.
| Cause: | Solution: |
|---|---|
| Primary antibody was not incubated at the correct dilution or was in the incorrect buffer. | Incubate primary antibody overnight at 4°C in TBS/T at the recommended dilution with the appropriate blocking agent. Polyclonal and rabbit monoclonal antibodies give the highest signal/noise in 5% BSA. Mouse monoclonal antibodies give the highest signal/noise in 5% milk. For individual antibodies please consult their respective datasheets for recommended dilution buffer. |
| Membrane is inappropriate for chemiluminescent detection. | Use only high quality nitrocellulose or PVDF membranes. Select a membrane found to exhibit high specific binding with low background. None of the nylon membranes tested were adequate for Western blotting. |
| Membrane blocking is not sufficient to prevent nonspecific binding of primary or secondary antibody. | Block for 1 hour at room temperature in 5% milk in TBS/T. Do not block excessively. |
| Secondary antibody was poor, incubated at the incorrect dilution, or in the incorrect buffer. | Some secondary antibodies bind nonspecifically to proteins in cell extracts. To assess the quality of a secondary antibody, perform a blot (through to film exposure) without primary antibody to determine whether this is a problem. Also, perform serial dilutions of the secondary antibody on blots with the same cell extracts and primary antibody to optimize concentration. Always incubate the secondary antibody in TBS/T, 5% milk for 1 hour in room temperature. |
| LumiGLO® reagent was prepared using poor quality water. | Chemiluminescent detection requires that high purity water be used to dilute LumiGLO® reagents A and B. Use only Milli-Q or other high quality water with organic and inorganic impurities removed. |
Problem: Low Signal
The protein of interest cannot be detected after 1–30 second exposure of blot to film.
| Cause: | Solution: |
|---|---|
| Insufficient incubation with primary antibody. | Phospho-antibodies generated against a single or dual phosphorylation site are highly specific but generally result in lower signal than regular antibodies. It is critical that these antibodies be incubated with blots overnight at 4°C in the appropriate buffer. |
| Incomplete transfer of proteins to membrane. | Transfer longer or at higher voltage. Monitor transfer using prestained molecular weight markers (membrane staining can inhibit chemiluminescent detection). |
| Blocking was excessive. | Blocking the membrane for too long in milk can obscure antigenic epitopes and strip proteins from the blot. Block for only 1 hour (never overnight). |
| Protein of interest is below detectable levels. | 20 µg total protein per lane is usually sufficient; more protein can be loaded if constitutive levels of the protein of interest are low. Load more sample or immunoprecipitate before SDS-PAGE. Try other cell lines or tissue sources where protein is more abundant. |
| Secondary antibody was weak. | Replace secondary antibody or increase the concentration in that step. |
| LumiGLO® reagent was improperly prepared or stock reagents A and B have become cross-contaminated. | Remake LumiGLO® reagent. Biotinylated molecular weight standards detected with anti-biotin-HRP act as positive controls for chemiluminescent detection. |
posted June 2005
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