A silver staining procedure for proteins in agarose gels

We modified the silver staining method of Kerenyi and Gallyas (Clin Chim Acta 1972; 38: 465-467) for visualizing low levels of protein with our own agarose gels as reported previously (Yokomizo K. et al. J Electrophoresis 2003; 47: 91-97). To reduce background staining and avoid the formation of artifact spots, a number of factors were studied. These included the composition of the staining reagent and fixative solutions (first and second fixation). It was found that 2.5% Na2CO3, 0.1% AgNO3, 0.1% NH4NO3, 0.75% tungtosilicic acid and 0.14% formaldehyde of the staining reagent mixture was critical and that the addition of 5% ZnSO4 to the second fixative solution resulted in consistently lower background staining and a significant reduction in artifact spots. Our silver staining procedure is approximately 100-fold more sensitive than Coomassie brilliant blue and the detection limit of bovine serum albumin is about 1.46 ng per band.