Estimation of Molecular Weight by Gel Filtration and Gel Electrophoresis

TLDR: This statistical analysis is concerned with the precision and accuracy of the standard calibration curve for estimation of molecular weights in gel filtration and PAGE in the absence of SDS.

Abstract: Gel filtration (GF) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) are perhaps the most popular methods for estimation of protein molecular weights. However, the precision and accuracy of these methods have never been rigorously evaluated by formal statistical methods. In contrast, a detailed objective statistical analysis has been applied to PAGE in the absence of SDS (Rodbard and Chrambach, 1970, 1974). This statistical analysis is concerned with the following: a. What is the precision of the “raw” measurement, e.g., elution volume (V e ), partition coefficient (K av), mobility (M), or relative mobility (Rf)? b. What are the precision and accuracy of the standard calibration curve for estimation of molecular weights? This includes several questions : What is the dependent variable? Is the calibration curve linear? Are there any outliers? What is the appropriate model? Is “ weighting” necessary, and, if so, how is it to be done? c. What are the standard error and 95% confidence limits for the molecular radius (or molecular weight) for the “ unknown” protein?