Western blot | Ponceau S Staining

Lab. of Bioclock & Aging/Dewan Md. Sumsuzzman

Ponceau S Staining

Ponceau S Staining Solution:

Table-1: Recipe of 0.1%  (w/v) Ponceau S in 5% (v/v) Acetic acid

Ingredients	Quantity
Ponceau S	1 gm
Acetic acid	50 ml
ddH2O	Make up to 1L

Table-2: Recipe of 0.2%  (w/v) Ponceau S in 3% (v/v) Acetic acid

Ingredients	Quantity
Ponceau S	2 gm
Acetic acid	30 ml
ddH2O	Make up to 1L

Table-3: Recipe of 0.1N NaOH in 100 ml water

Ingredients	Quantity
NaOH	0.4 gm
H2O	100 ml

Protocol:

1. Following Western blotting, transfer the membrane to 5ml Ponceau S Stain solution.
2. Place on an orbital shaker for 5 minutes at room temperature.
3. Rinse membrane with DI water to achieve desired staining, approximately 2‐3 washes of 5 minutes each will remove the background staining.
4. For total protein destaining, use a 0.1N NaOH solution. Wash the membrane with 0.1N NaOH solution for 5 minutes. Protein bands will start disappear after 10-30 seconds. Discard wash solution and repeat once.
5. Wash the membrane 2‐3 times with DI water for 5 minutes each.
6. Continue with Western blot/ immunoblot procedure.

Note:

• The product can be used on most membranes including PVDF (polyvinylidene difluoride) except nylon membranes. Ponceau S does not destain from nylon membranes because the nylon is positively charged, the stain is negatively charged, and therefore, the stain is electrostatically held. Other membranes, like nitrocellulose, cellulose acetate, or PVDF are electrically neutral, and therefore, can easily be destained.

• A quick water rinse allows one to see if bands are developing, but destaining is usually done in 10% acetic acid. For sequencing or immunoassay purposes, the bands can be destained to colorless using 0.1 M NaOH.

• If using nitrocellulose membranes, do not use alcohols in destaining procedure. For a western blot, rinsing the membrane with deionized water will remove any background dye. To remove the dye from the protein for further immunochemical testing, a rinse the membrane with 0.1 M NaOH. Destaining in acetic acid is not done on the nitrocellulose membrane.