Experimentally improved reliability of ultrasensitive silver staining of protein in polyacrylamide gels

Variations of the ultrasensitive silver staining method of B. R. Oakley, D. R. Kirsch, and N. R. Morris ( Anal. Biochem. 105, 361–363 (1980)) have been tested. It was established that the reliability of the method was greatly improved if (i) free silver was carefully washed out before reduction with formaldehyde; (ii) the extent of development was controlled by using methylamine to inactivate the formaldehyde; and (iii) the optimum quantity of ammonia, which was found to be 4 mol/mol of silver was used (this quantity was defined as that which titrates a particular amount of 1 n HCl). The time of preparation of the formaldehyde reducer was found not to be highly critical. In our hands the method can detect down to 0.1 ng of protein/5-mm slot.