An agarose gel electrophoresis method for the separation of arabinogalactan proteins

A method for resolving plasma membrane associated arabinogalactan proteins (AGPs) has been developed. Plasma membranes purified by aqueous polymer two-phase partitioning were first subjected to Triton X-114 fractionation. The resulting water phase contained all detectable plasma membrane-bound AGPs. Plasma membrane AGPs were then resolved in an SDS-agarose gel electrophoresis system (SDS-AGE). For separating plasma membrane AGP species of the same apparent molecular weight but with different net charge, a two-dimensional electrophoresis system was used, utilizing isoelectric focusing in an immobilized pH gradient in the first dimension and SDS-AGE in the second dimension. These methods enabled the separation of individual plasma membrane AGPs. In comparison, SDS-PAGE methods left AGPs as unresolved high molecular-weight smears. The methods described here may help to establish some basic features of AGPs, such as the number, organization, and protein and carbohydrate characteristics of plasma membrane AGPs, as well as the relationship between plasma membrane and extracellular AGPs.