Electrophoresis in Highly Cross-linked Polyacrylamide Gels

Abstract

The properties of polyacrylamide gels with 1 to 50% cross-linking (%C) were studied using electrophoresis in multiphasic buffer systems of five proteins with known molecular weight. The extrapolated free mobility (Y ₀ or M ₀ varies in a biphasic manner with %C, reaching a maximum when %C is 5 to 7%, and falling to a constant plateau value specific for each protein when %C is greater than 20%. The retardation coefficient (K_R ) shows a different type of biphasic relationship with %C: it increases with %C over the range 1-5%C and then falls asymptotically to zero, irrespective of molecular size. High %C provides a substantial increase in the effective pore size of the gel and enables one to fractionate macromolecules in the multimillion molecular weight range. These gels should be useful when it is desired to minimize molecular sieving, e.g., for isotachophoresis and isoelectric focusing.