SDS-PAGE is not a reliable method for accurately determining the molecular weight of proteins. This is why the term apparent molecular weight is often used. The molecular weight estimated by SDS-PAGE can be very different from the actual molecular weight. Matagne et al. reported 55 kDa for a 29-kDa b-lactamase (1), whereas the apparent weight of a 114-kDa protein was measured as 180 kDa (2). Separation of proteins by SDS-PAGE depends on the uniform binding of negatively charged SDS to the protein to give a constant charge-to-mass ratio, but in reality the amount of SDS bound can vary for different proteins. Many factors can affect mobility on SDS polyacrylamide gels, such as hydrophobicity, charge, glycosylation (for proteins expressed in eukaryotic expression systems), and phosphorylation (3). In some proteins, even substitution of single amino acids has been reported to result in altered mobility (4). Also glycolysation and phosphorylation e.g. in insect cells can cause altered mobility
1. Matagne, A., Joris, B., and Frere, J.-M. (1991) Anomalous behaviour of a protein during SDS/PAGE corrected chemical modifcation of carboxylic groups. Biochem. J. 280, 553.
2. Casaregola, S., Jacq, A., Laoudj, J., McGurk, G., Margarson, S., Tempete, M., Norris, V., and Holland, Cloning and analysis of the entire Escherichia coli ams gene. J. Mol. Biol. 228, 30.
3. Hames, B.D. (1990) One-dimensional polyacrylamide gel electrophoresis. In: Hames, B.D. and Rickwood,
Gel electrophoresis of proteins: a practical approach. Oxford: Oxford University Press, p 1.
4. Fasano, O., Alfdrich, T., Tamanoi, F., Taparowsky, E., Furth, M., and Wigler, M. (1984) Analysis of the potential of the human H-ras gene by random mutagenesis. Proc. Natl. Acad. Sci. USA 81, 4008.
Hi. I'm blotting for a membrane channel... I'm using 12% SDS...
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