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