Careful attention to your handling and protocol steps is required, and multiple trials may be necessary to resolve this problem. If you observe high background across the blot, there are a number of likely causes. See Blotting Membranes and Papers to learn more about the different types available and their properties If the membrane dries, rewet in methanol and re-equilibrate in TBS-T (caution: may adversely affect downstream detection processes) Once wet, do not allow membrane to dry out. Because of the hydrophobic nature of PVDF, the membrane must be prewetted in methanol prior to equilibration in aqueous transfer buffer. PVDF: White regions on PVDF membrane indicate areas where the membrane was either inappropriately prewetted or allowed to dry out.Equilibrate in transfer buffer until ready to use If wetting does not occur immediately by immersion of the sheet in transfer buffer, heat distilled water until just under boiling point and soak the membrane until completely wet. Nitrocellulose: White regions on the nitrocellulose membrane indicate dry areas where protein will not bind.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 guide