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Home Protein Troubleshooting Guide: Western Blotting

Troubleshooting Guide: Western Blotting

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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.



Last Updated ( Sunday, 05 October 2008 04:52 )