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Protein staining and analysis of 2D gels

Protein staining and analysis of 2D gels

Gels were stained with either silver nitrate according to a modified procedure of Blum et al. (1987) or the GelCode blue stain reagent from Pierce (Rockford, IL, USA), using the Hoefer® Automated Gel Stainer apparatus from Amersham Pharmacia Biotech. Silver stained gels were scanned with the Sharp JX-330 scanner equipped with the Labscan version 3.00 from Amersham Pharmacia Biotech. Image analysis was carried out with the ImageMaster 2-D Elite version 3.01 software (Amersham Pharmacia Biotech), according to the instruction booklet “ImageMaster® 2D Elite” from Amersham Pharmacia Biotech. After spot detection and background substraction (mode: average on boundary), 2D gels were aligned, matched, and the quantitative determination of the spot volumes was performed (mode: total spot volume normalization). For each analysis, statistical data showed a high level of reproducibility between normalized spot volumes of gels produced in triplicate from the two independent protein extractions.

  • Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93-99

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