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04-0350 Mol Weight tn.FIN3 - Bio-Rad Laboratories

IntroductionSodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) is a reliable method for determining the molecularweight (MW) of an unknown protein. The first step in MWdetermination of a protein is to separate the protein sample on the same gel with a set of MW standards. Next, a graph of log MW vs. relative migration distance (Rf) is plotted, basedon the values obtained for the bands in the MW standard. The MW of the unknown protein band is then calculated byinterpolation using this graph. The key to determining MWaccurately is selecting separation conditions that will producea linear relationship between log MW and migration within thelikely MW range of the unknown Conditions for MW DeterminationTo ensure accurate MW determination, both the proteinstandards and the unknown protein must be electrophoresedon the same gel under identical separation conditions.

the data points that lie beyond the linear range, as shown in Figure 3. In Figure 3B, the values for the five largest proteins were omitted from the calculations, increasing the r2 value from 0.913 to 0.997. Most scientific calculators and software can generate the r2 value and the equation of the best-fit line from the data points.

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