Ponceau S Staining Solution  is a rapid and reversible stain for detecting protein bands on Western blot membranes and can be used with PVDF, nitrocellulose and cellulose acetate membranes.

Microgram quantities of transferred protein can be detected with Ponceau‐S Stain, which generates reddish pink protein bands with a clear background.
The limit of detection for this stain is 100 ng BSA/ band transferred to nitrocellulose membranes.

Ponceau S is a negative dye that binds to the positively charged amino acids.

This staining technique is reversible to allow further immunological detection. The limit of detection for this stain is 250 nanograms of protein after separation by electrophoresis in polyacrylamide gels and transferred to nitrocellulose membranes. Ponceau S is a negative stain which binds to the positively charged amino groups of the protein. It also binds non-covalently to non-polar regions in the protein. (Note: Ponceau S is not suitable for use with nylon membranes.)

The stain is useful because it does not have a deleterious effect on the sequencing of blotted polypeptides and the user can completely remove it from the protein bands by continued washing.
 
Procedure for total protein detection:
1. After electrophoresis, immerse the blotted membrane in a sufficient amount of Ponceau S Staining Solution and stain for 5 minutes. Do this before blocking.
2. After staining, immerse the membrane in an aqueous solution containing 5% acetic acid (v/v) for 5 minutes, change the aqueous solution, and immerse the membrane for another 5 minutes.
3. Then, transfer the membrane into water for two washes of 5 minutes each.
4. Finally, remove the membrane and block as normal.