Premium
A new silver staining apparatus and procedure for matrix‐assisted laser desorption/ionization‐time of flight analysis of proteins after two‐dimensional electrophoresis
Author(s) -
Sinha Pranav,
Poland Julia,
Schnölzer Martina,
Rabilloud Thierry
Publication year - 2001
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200107)1:7<835::aid-prot835>3.0.co;2-2
Subject(s) - silver nitrate , chemistry , staining , chromatography , tetrathionate , distilled water , silver stain , potassium carbonate , desorption , matrix assisted laser desorption/ionization , stain , matrix (chemical analysis) , pipette , mass spectrometry , potassium , nuclear chemistry , adsorption , microbiology and biotechnology , organic chemistry , medicine , thiosulfate , sulfur , pathology , biology
We report on a new silver stain especially developed for staining large gels (25 cm× 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 νg per gel.