Staining the blot with Ponceau S

Ponceau S can be used to verify transfer and to locate molecular weight markers on nitrocellulose membrane. The stain is not very sensitive; the staining is rapid but the red stain will wash away in subsequent washes.

 

Staining solution:

0.2 Ponceau S (3-hydroxy-4-[2-sulfo-4-(sulfo-phenylazo)phenylazo]-2,7-naphthalene disulfonic acid) 200 mg

3 trichloroacetic acid (TCA) 3 g

add H2O to 100 ml.

The solution is stable at room temperature for >1 year.

  • Incubate the nitrocellulose membrane in the stain for 2-5 min at room temperature with agitation.

  • Pour the stain back into the bottle (it can be reused several times!) and wash the blot with water until the bands/spots become visible. Mark molecular weight markers and position of transfer as required.

  • Wash the blot in TBS or PBS and proceed to the blocking

 

Staining the blot with Colloidal Gold

Colloidal gold protein stain (’Bio-Rad’, No. 170-6527) can be used to verify transfer on nitrocellulose and PVDF membranes. It binds to all proteins on the blot and stains them dark red. Detection sensitivity is about 1 ng; the staining is slow and irreversible.

The stain is usually used after the development of the blot.

  • Incubate the membrane in the stain for 20-30 min at room temperature with agitation until the bands/spots become visible.

  • Wash the blot with water and dry.

 

Staining the blot with Coomassie Blue

Coomassie blue is used to locate proteins for the N-terminal sequencing on PVDF membrane. It binds to all proteins on the blot and stains them blue. Detection sensitivity is about 80 ng.

  • Incubate the membrane in the stain for 2-5 min at room temperature with agitation until the spots become visible.

  • Wash the blot with water and dry.

 

To reduce the background, incubate the blot in a standard destaining solution, used to destain Coomassie stained gels.

 

2004 02 12 – Julius