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.
To ensure transfer is complete, stain the membrane with Ponceau S, Amido Black or India Ink.
Use prestained MW marker.
Possible source: Over transfer
Suggestion: Reduce voltage or time of transfer for low molecular weight proteins (< 10 kDa).
Possible source: Isoelectric point is >9
Suggestion: Use alternative buffer system with higher pH such as CAPS (pH 10.5).
Possible source: Incorrect secondary antibody used
Suggestion: Confirm host species and Ig type of primary antibody.
Possible source: Old antibody
Suggestion: If antibody is expired or past manufacturer warranty, purchase fresh antibody.
Possible source: Incorrect storage of antibodies
Suggestion: Follow manufacturer's recommended storage and avoid freeze/thaw cycles.
Possible source: Sodium Azide contamination
Suggestion: Make sure buffers do not contain Sodium Azide as this can quench HRP signal.
Possible source: Insufficient incubation time with primary antibody
Suggestion: Extend incubation time to overnight at 4°C.