Agarose Gel Electrophoresis of High Molecular Mass Protein Complexes

摘要

Polyacrylamide gel electrophoresis has long been the method of choice forseparating native and denatured proteins. Depending upon the concentration of polyacrylamide, these gels can typically resolve proteins with a mass of up to 200 kDa; in extreme instances, concave exponential gradients of 3%-20% polyacrylamide have been used to resolve proteins in the range of 12-1000 kDa. Electrophoretic separation of larger proteins and protein complexes requires the larger pore size of agarose gels. For example, aggregating proteoglycans and multimeric forms of von Willebrand factor have been achieved using vertical agarose gels discontinuous buffer systems. Larval salivary glands of the midge, Chironomus tentans synthesize three size classes of silk proteins, the largest of which, the spls, range from 1000-1500 kDa (for review, see Reference 3). The masses of these proteins have been estimated by biochemical techniques (5, 6, 12) and confirmed by molecular biological cloning of their genes. The spIs form the fibrous backbone of insoluble silk threads; however, soluble spIs can be extracted from the lumen of salivary glands at an intermediate step of assembly. They can be disassembled at this step and reassembled in vitro into protein complexes which, by electron microscopy, appear as networks of fibrils and beaded fibers.