Question

Ponceau S is a red dye that reversibly binds to all proteins. We will use this stain prior to developing the Western blot next week with antibodies. Assume that you do not observe any protein bands on the nitrocellulose blot after performing the Ponceau S stain. Describe one likely procedural error during the transfer set-up that would account for this observation.

Answer 1

Ponceau S is a stain that is used to stain proteins. Staining with Ponceau S is fast, simple, and easily reversible. With the help of this stain, the protein transferred on the nitrocellulose blot appears as reddish-pink bands on the pink-colored background.

No observation of protein bands on the nitrocellulose blot even after performing Ponceau S staining may be due to the procedural error wherein no transfer of protein would have occurred during Western blotting. This occurs when the placement of the nitrocellulose membrane and the SDS-PAGE gel (sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel) is reversed. This causes the protein to transfer to the filter paper (placed in contact with the gel) instead of the nitrocellulose membrane. Thus, the Ponceau S could not detect the protein as they are absent.

This procedural error can be presented by carefully checking that the nitrocellulose membrane is on the right side of the SDA-PAGE gel. The proper contact between the gel and membrane is crucial during blotting.