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Protocols, optimization tips,
troubleshooting guides,
and more for Western Blot.
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Western Blot Troubleshooting: Weak Signal
A weak western blot signal is characterized by faint or indistinct bands. While the bands may be barely visible at their predicted sizes, weak signal can often require repeating the experiment. Common sources of this error occur during protein transfer and detection. Use these tips to identify the source of the error and get better results:
| Transfer issues | Insufficient sample concentration | Increase the amount of starting material |
| Concentrate your sample using immunoprecipitation or similar procedure | ||
| Transfer too vigorous | Reduce transfer time or voltage to prevent small proteins transferring completely through membrane | |
| Use a secondary membrane to capture proteins transferred through the primary membrane | ||
| Use a membrane with smaller pore size | ||
| Inadequate transfer | Increase transfer time or voltage | |
| Sandwich assembly oriented incorrectly | Make sure the sandwich assembly is oriented correctly relative to the electric field | |
| Check the polarity of the electric field | ||
| Incorrect transfer buffer PH | Adjust transfer buffer PH to be 2 points lower than the pI of protein sample to optimize charge:mass ratio | |
| Detection issues | Insufficient antibody concentration | Use a dot blot assay to optimize protein concentration |
| Insufficient antibody binding affinity | Reduce washing stringency | |
| Increase antibody concentration | ||
| Use Boster high affinity primary antibodies | ||
| Insufficient sample loading | Use more starting material | |
| Concentrate sample prior to loading | ||
| Antigens masked by dry milk blocking solution | Nonfat dry milk can sometimes mask antigens. Try using a different blocking reagent |
Keywords: Western Blot, Troubleshooting, Weak Signal, Low Staining, Faint Bands
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