Silver-enhanced copper staining of protein blots

Copper staining of protein blots has previously been shown to detect proteins rapidly, quantitatively, and sensitively. The sensitivity of this assay can now be enhanced nearly 10-fold with an additional step of silver staining. The silver-enhanced assay retains many of the virtues of copper staining, including a quantitative optical response with the amount of adsorbed protein, low variation between proteins, and reversibility. In addition, both copper staining and silver-enhanced copper staining do not detect nucleic acid, thus allowing for selective measurement of nucleic acid binding proteins or protein contamination in nucleic acid preparations. Silver-enhancement requires only a 5-min incubation with a single reagent. Since copper staining is performed first, it can be used to determine if the greater sensitivity provided by silver enhancement is desirable. For quantitation, an inexpensive reflectance scanning densitometer utilizing optical scanners and NIH Image is described.