A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis.
Silver staining has been the method most commonly employed for high sensitivity staining of proteins following two-dimensional gel electrophoresis. Whilst this method offers detection in the nanogram range it does have major drawbacks including a lack of linearity, nonstoichiometric staining of proteins, a lack of compatibility with the microchemical preparation of proteins for identification by mass spectrometric techniques, and a highly subjective assessment of the staining endpoint. SYPRO Ruby is a relatively new, ruthenium complex-based stain which is reported to offer advantages over silver, particularly in overcoming the limitations cited above. We describe a series of experiments where several protein staining procedures commonly employed are compared. To enable optimization of the in situ digestion procedure, a statistical approach has been undertaken. The effects of a variety of staining, digestion, and analysis protocols on the downstream processing of a test radiolabeled protein were studied. The data confirms that as well as offering sensitivity similar to silver, SYPRO Ruby staining is reproducible, linear, and offers a higher level of compatibility with the identification of proteins by mass spectrometry.
White
Ian R
IR
Genomic and Proteomic Sciences, GlaxoSmithKline Pharmaceuticals, Stevenage, UK.
Pickford
Russell
R
Wood
John
J
Skehel
J Mark
JM
Gangadharan
Bevan
B
Cutler
Paul
P
D004396:Coloring Agents; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D015233:Models, Statistical; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D020543:Proteome; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004396:Coloring Agents; D010455:Peptides; D011506:Proteins; D020543:Proteome