A colloidal silver staining--destaining method for precise assignment of immunoreactive spots in two-dimensional protein patterns.
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.
Vettermann
Christian
C
Division of Molecular Immunology, Department of Internal Medicine III, Nikolaus Fiebiger Center for Molecular Medicine, University of Erlangen-Nürnberg, 91054 Erlangen, Germany.
Jäck
Hans-Martin
HM
Mielenz
Dirk
D
D000818:Animals; D003102:Colloids; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D051379:Mice; D010449:Peptide Mapping / Q000379:methods; D011506:Proteins / Q000032:analysis*; D012834:Silver / Q000737:chemistry*; D016622:Silver Staining / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003102:Colloids; D011506:Proteins; D012834:Silver