western blot troubleshooting high background

Use a cooling coil or “blue ice” insert in the cellĬooling for Bio-Rad Wet Tank Blotting Systems:ĭried out or improperly hydrated membrane.Perform transfer in tank apparatus placed in ice or a temperature-controlled (4☌) room.Reduce current, and increase transfer time to compensate.High-intensity transfers in tank blotting sample can cause buffer heating, leading to bubble formation ensure that buffer remains cool.Visualize total protein on gels and blots using Bio-Rad’s Stain-Free Gels featuring our proprietary Stain-Free Technology).

To verify protein transfer, stain the membrane with Ponceau S after blotting.To determine if there is residual, untransferred protein remaining on the gel, use a total protein stain on the gel after transfer, e.g., Coomassie, colloidal gold, or SYPRO Ruby.For example, Coomassie and colloidal gold are not compatible with downstream steps (see Bio-Rad Protein Stains and the Protein Stain Selection Guide). If planning to use the blot in downstream steps, make sure that your stain can be removed or is compatible with antibody detection. When using a tank blotter, check the quality of the sponges (foam pads) if a snug assembly can no longer be made between the frames, the pads should be replacedįoam pads for Bio-Rad wet tank blotting systems:Īs a diagnostic test, you can check transfer quality by imaging proteins on the gel and blot.Consider using a rapid transfer pack with the Trans-Blot ® Turbo™ Rapid Blotting System to help prevent improper sandwich assembly.Ensure all filter papers and sponges used in assembly are properly saturated and equilibrated with transfer buffer Use more buffer when assembling transfer sandwich.Carefully remove air bubbles between the gel and the membrane before protein transfer using a roller or glass rod.Improper assembly of transfer sandwich, trapped air bubbles White spots or white areas on the blot may be due to trapped air bubbles or unwetted membrane areas where protein will not bind.

Western blot troubleshooting high background