Quantification of silver-stained proteins resolved by two-dimensional electrophoresis: genetic variability as related...

Relative abundance and solubility of proteins from etiolated coleoptiles of maize were investigated using two-dimensional electrophoresis (2-D PAGE). Automatic quantification of silver-stained polypeptides on replicate 2-D gels made it possible to test the linearity of the relationship between spot integrated optical density and protein amount in the range from 15 micrograms to 135 micrograms per gel, in two inbred lines. A linear response was found for more than 60% of the spots in each genotype. When a linear response was found in both lines for a given spot, the slope values were similar in 94% of the cases, indicating the reliability of silver staining for polypeptide quantification. The parameters of the curves allowed the definition of protein classes of different abundances that could be compared for genetic variability between the two lines. From a comparison between standard 2-D patterns (trichloroacetic acid-acetone extracted proteins) and patterns obtained from Tris buffer extracted proteins, it appeared that 92% of the proteins visualized in the standard gels were soluble. No difference in genetic variability, either qualitative or quantitative, was evidenced between the various classes of abundance, or between soluble and insoluble proteins.