A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis.
We report on a new silver stain especially developed for staining large gels (25 cm x 20 cm) from the Hoefer ISO-DALT system for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI-TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch-to-batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI-TOF can be performed with protein loads as little as 100 micrograms per gel.
Sinha
P
P
Institut für Laboratoriumsmedizin und Pathobiochemie, Universitätsklinikum Charité, Campus Charité Mitte, Schumannstrasse 20-21, 10117 Berlin, Germany. pranav.sinha@charite.de
Poland
J
J
Schnölzer
M
M
Rabilloud
T
T
D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D006801:Humans; D007633:Keratins / Q000302:isolation & purification; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000302:isolation & purification; D000091402:Prohibitins; D011506:Proteins / Q000302:isolation & purification*; D020543:Proteome / Q000302:isolation & purification; D012097:Repressor Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012834:Silver; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013194:Staining and Labeling / Q000295:instrumentation* / Q000379:methods* / Q000706:statistics & numerical data; D022021:Tacrolimus Binding Proteins / Q000302:isolation & purification
D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D000091402:Prohibitins; D011506:Proteins; D020543:Proteome; D012097:Repressor Proteins; D012834:Silver; D007633:Keratins; D022021:Tacrolimus Binding Proteins