Acrylamide Polymerization A Practical Approach ( l porosity. Gel E...)

Acrylamide Polymerization A Practical Approach

l porosity.

Gel Exclusion Limit Determination
Estimation of protein molecular weight by SDS-PAGE is a widely employed procedure. The relative mobility of a protein in an SDS-PAGE gel is related to its molecular weight. A standard curve is constructed with proteins of known molecular weight by plotting the logarithms of their molecular weights versus the relative mobilities of the proteins. The relative mobility of a protein of unknown molecular weight is then fitted to the curve to determine its molecular weight.

A standard curve can be extrapolated to give the y-intercept, which represents the molecular weight exclusion limit of that particular gel. That is, proteins with a molecular weight greater than the y-intercept value will show zero mobility and will be excluded from the gel matrix.

Poorly polymerized gels have greater porosity due to incomplete chain elongation and crosslinking. As a result, the exclusion limit will be greater than for a well-polymerized gel of the same percent acrylamide. Furthermore, when polymerization is incomplete, exclusion limits are irreproducible. Use of highly purified gel-forming reagents and proper polymerization technique will result in the lowest and most reproducible exclusion limits for a given percent total monomer.

Figure 2 shows a typical curve obtained by plotting log molecular weight versus relative mobility following SDS-PAGE for a group of standard proteins. The antilog of the y-intercept value of this plot is 115,000 as determined by linear regression analysis. The approximate molecular weight exclusion limit of the gel is thus 115,000. The y-intercept value should be considered approximate because it de
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( l porosity. Gel E...)Acrylamide Polymerization A Practical Approach