This is a much more detailed troubleshooting for western blotting. We hope it will be helpful to biological researcher, especialy the beginners.

Problem: No Bands Observed

Possible source: Insufficient antibody

Suggestion: Antibody may have low affinity to protein of interest. Increase antibody concentration (2-4 fold higher than recommended starting concentration).

Antibody may have lost activity. Perform a Dot Blot.

Possible source: Insufficient protein

Suggestion: Increase the amount of total protein loaded on gel.

Confirm the presence of protein by another method.

Use a positive control (recombinant protein, cell line or treat cells to express analyte of interest).

Perform a Dot Blot.

Possible source: Poor transfer

Suggestion: Wet PVDF/Immobilon-P membrane in methanol or nitrocellulose membrane in transfer buffer.

Ensure that there is good contact between PVDF membrane and gel.

Possible source: Incomplete transfer

Suggestion: Optimize transfer time. High MW protein may require more time for transfer.