Immunological detection of proteins requires that proteins be transferred and immobilized onto a membrane support after electrophoresis (see Western Blotting). Staining of the immobilized proteins establishes transfer efficiency, and allows the operator to mark the membrane with the locations of lanes and size markers, facilitating later analysis. The mechanism of staining is the same as for in-gel staining. Conditions must be established under which the dye binds more avidly to the protein than to the support, resulting in dark, high contrast zones corresponding to the presence of protein. However, the requirements for the staining are different. Sensitivity is less of an issue, as markers are generally loaded in high concentration, and lanes of sample will show up even when individual bands in the lanes may be faint. Speed and protein recovery (not stripping the protein from the membrane) are more important in this case.

Staining of Proteins Immobilized on Membranes

Ponceau S

1. Mix a stock solution of Ponceau S.  To make a stock solution of Ponceau S, dissolve 0.5g Ponceau S in 100 ml of 1% aqueous acetic acid.
2. Immerse blot in Ponceau S stock solution for 5 minutes.
3. Transfer blot to deionized H₂O, and agitate until bands appear (1-5 minutes).
4. Mark bands with indelible ink (note: bands will fade in 15 minutes).

India Ink

1. Wash blot in 0.3% Tween 20 in phosphate-buffered saline (PBS), five times for 30 minutes each at 37°C.
2. Transfer blot to India Ink solution (0.1% Pelikan 17 Black or equivalent in 0.3% Tween 20 PBS)
3. Rinse membrane 2 times in Tween solution. Destain in Tween 20 solution until desired contrast is achieved. This protocol will detect as little as 50 µg per band.

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