A colloidal silver staining--destaining method for precise assignment of immunoreactive spots in two-dimensional protein patterns.

Abstract

The characterization of protein expression patterns by two-dimensional gel electrophoresis depends on efficient and reliable identification strategies for target spots. In addition to sophisticated techniques, such as microsequencing and peptide mass spectrometry, immunodetection of membrane-immobilized proteins is a valuable method with which to identify the corresponding spots for a given set of candidate proteins. To precisely assign immunoreactive spots, this approach requires specific immunodetection and staining of total protein to be performed on the same membrane. Here, we describe a highly sensitive, colloidal silver-based method for the assignment of immunoreactive spots in two-dimensional protein patterns. This simple and rapid procedure involves a destaining step after staining of nitrocellulose-bound proteins with colloidal silver. We show that destaining of proteins is a prerequisite for subsequent immunodetection using enhanced chemiluminescence. Several types of antibodies were successfully employed for antigen detection after the staining-destaining procedure. Our results demonstrate that the colloidal silver-based method is generally applicable for the unambiguous identification of candidate proteins in complex two-dimensional patterns.