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Star Republic: Guide for Biologists

Staining of proteins

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Proteins in a polyamcriamide gel or transfered on a membrane can be stained with a number of dyes (e.g., Commassie blue and Ponceau S).

See Coomassie blue staining of proteins for staining a polyamcriamide gel. The following protocols can be used for staining proteins transfer onto PVDF or nitorcellulos membranes.

Coomassie blue staining of membrane

1. After the proteins have been transfered onto the membrane, rinse the membrane with dH2O.

2. Saturate the membrane with 100% methanol for a few seconds.

3. Stain with 0.1% Coomassie Blue 250 in 40% methanol-1% acetic acid for 1-5 minutes.

4. Destain 1 to 3 times with 50% methanol-1% acetic acid (about 15 minutes total).

5. Wash with dH2O.

Ponceau S staining of membrane

1. After the proteins have been transfered onto the membrane, rinse the membrane with dH2O.

2. Stain with 0.2% Ponceau S in 1% acetic acid for 5 minutes.

3. Destain 1 to 3 times with 5% acetic acid.

4. Wash with dH2O.

Amido Black (Napthol Blue Black) staining of membrane

1. After the proteins have been transfered onto the membrane, rinse the membrane with dH2O.

2. Stain with 0.1% Amido Black in 50% methanol-10% acetic acid for 5 minutes.

3. Destain 1 to 3 times with 20% methanol-7.5% acetic acid.

4. Wash with dH2O.

India Ink (Pelikan, Fount India Black) staining of membrane

1. After the proteins have been transfered onto the membrane, rinse the membrane with dH2O.

2. Wet membrane briefly in 100% methanol, then incubate briefly in TBST (25 mM Tris, 140 mM NaCl, 3 mM KCl, 0.05% Tween-20, pH 8.0).

3. Stain with 0.1% India Ink in TBST for 5 minutes.

4. Destain with several changes of TBST.