A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis
| Autor: | Martina Schnölzer, Thierry Rabilloud, Pranav Sinha, Julia Poland |
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| Rok vydání: | 2001 |
| Předmět: |
Silver
Proteome Potassium HSP27 Heat-Shock Proteins chemistry.chemical_element Mass spectrometry Sensitivity and Specificity Biochemistry Cell Line Tacrolimus Binding Proteins Silver stain Potassium carbonate chemistry.chemical_compound Prohibitins Humans Electrophoresis Gel Two-Dimensional Molecular Biology Heat-Shock Proteins Tetrathionate Chromatography Staining and Labeling Proteins Reproducibility of Results Neoplasm Proteins Staining Repressor Proteins Silver nitrate chemistry Distilled water Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Keratins Molecular Chaperones |
| Zdroj: | PROTEOMICS. 1:835-840 |
| ISSN: | 1615-9861 1615-9853 |
| DOI: | 10.1002/1615-9861(200107)1:7<835::aid-prot835>3.0.co;2-2 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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