Glycogen size analysis by mini-gradients

Abstract

The separation of low (cytosolic) and high (lysosomal) molecular weight glycogen on small induced citrate gradients has been compared with the conventional sucrose gradient separation. The citrate method is shown to be vastly more sensitive and can be used with as little as 0.1 mg of glycogen, compared with 8 mg which is the normal loading on a sucrose gradient. The citrate method has been tested with fractionated and degraded glycogen samples and found to give satisfactory results, although with higher degrees of error, when compared with the conventional method. The success of the citrate method arises from the previously undescribed aggregation of high molecular weight glycogen in the presence of 1.09 M sodium citrate, which has been measured by laser dynamic light-scattering. This phenomenon allows satisfactory separation of the variously sized glycogens to be achieved with centrifugal forces of ca. one-tenth of those used in sucrose density gradient analysis, thereby considerably simplifying the separation methodology.