A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis

Abstract

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.

The Depressive Phenotype Induced in Adult Female Rats by Adolescent Exposure to THC is Associated with Cognitive Impairment and Altered Neuroplasticity in the Prefrontal Cortex

“…The second dimension was carried out on 45 mA/gel at 18°C. Gels were stained according to MS-compatible Sinha's silver staining method (Sinha et al 2001). After staining, gels were scanned with an EPSON V750 Pro transmission scanner and images were analyzed with ImageMaster 2D Platinum V5.0 software package (GE Healthcare).…”

Biochemical characterisation and antioxidant activity of mycelium of Ganoderma lucidum from Central Italy

“…The second dimension was carried out in a Laemmli system on 9-16% polyacrylamide linear gradient gels (18 cm  20 cm  1.5 mm) at 40 mA/gel constant current, until the dye front reached the bottom of the gel. Analytical gels were stained with silver nitrate (Sinha, Poland, Schnölzer, & Rabilloud, 2001). Gel images were acquired by Fluor-S MAX multi-imaging system (BioRad) and the data were analysed, including spots detection, quantification and normalisation, using ImageMaster 2D Platinum version 5.0 software (GE Healthcare), in particular, protein quantification values were calculated as relative volume (%).…”

Characterization of the differential protein expression associated with thermoresistance in human gastric carcinoma cell lines

“…Proteins were visualized after analytical (10 mg total protein extract) two-dimensional electrophoresis using the silver staining method performed according to Hochstrasser et al (http://www.expasy.ch/ch2d/protocols/ protocols.fm4.html) and according to Sinha et al [17] for MALDI-TOF-MS analysis (100 mg total protein extract) identification of proteins. Briefly, the gels were taken out of the glass cassettes and placed on the plexiglass grid of the staining apparatus.…”