Ponceau S Staining of Nitrocellulose Membrane after Western Blotting


Staining of nitrocellulose membrane using Ponceau S is often used to evaluate the quality and transfer efficiency of western blotting. After staining, all protein bands appear as pink color bands on the membrane, making it conveniently analyze the banding pattern of fractionated protein without the need for any sophisticated instruments. 

Since Ponceau S reversibly stains the protein, after checking the transfer, the membrane is destained by washing with TBST and is used for immunodetection.


Reagents and solutions
0.1% (w/v) solution of Ponceau S
Nitrocellulose membrane with proteins
Deionized/distilled water

Equipment and disposables
Container that can accommodate nitrocellulose membrane


Step 1: Remove the nitrocellulose membrane after transferring the protein from the polyacrylamide gel. Now keep the membrane in Ponceau staining solution submerged at room temperature.

Step 2: Shake gently for 5 min. Remove the stain and rinse the membrane with Distilled or Deionized water two times.

Note: Protein bands will be seen as red/pink color bands. Take a photograph for the record.

Step 3: Destain the membrane by washing with PBST/TBST several times until all pink color disappears. Usually, it takes 2 – 3 washings.

Now process the membrane for immunodetection.

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