western blot troubleshooting high background

Ensure that the buffer is cool by reducing the current and increasing transfer time to compensate

Tank blotting with high-intensity transfers can lead to heating the buffer and formation of bubbles: Consider that collodial gold and coomassie are not compatible with downstream steps. If you want to use the blot in downstreams steps, confirm that the stain can be removed or its compatibility with antibody antibody detection. Running a diagnostic test like checking transfer quality via imaging proteins on the gel and the blot is advisable. You can visualize total protein on blots or gels using stain-free gels. In order to be sure that the transfer is successful, staining the membrane with Ponceau S after blotting is advisable. Checking for residual protein or protein unable to transfer, remaining on the gel, using a total protein stain is advisable after transfer is complete.

If you use a tank blotter, make sure that the sponges(foam pads) can create a proper assembly and if they can no longer create a good seal, the sponges should be replaced with new ones. Using a rapid transfer pack greatly helps preventing improper sandwich assembly. Carefully remove all air bubbles present between the membrane and the gel prior to transferring the protein, either a glass rod or a roller can be used. Causes:įaulty assembly of transfer sandwich, air bubbles are present. Both can be easily prevented with improving your technique and additional care. White areas on WB background can be caused by air bubbles or dry areas of membrane restricting proteins from binding.

Shadow transferred on the blot (tank blotters).

During Western Blotting, several issues may occur with the background: In this article we discuss and provide solutions for common Western Blot background problems, but if you have any other issues with this analytical technique, please consider using our other articles about issues with signal strength, protein band size and pattern and band appearance. Troubleshooting issues with Western Blot background with St John's Laboratory Introduction:

Western blot troubleshooting high background