Separation and Identification of Native Proteoglycans by Composite Agarose-Polyacrylamide Gel Electrophoresis and Immunoblotting.

Composite agarose-polyacrylamide gel electrophoresis (CAPAGE) in gels of 1.2% w/v polyacrylamide and 0.6% w/v agarose can be used to examine the heterogeneity of full-length native proteoglycan populations and their fragments in crude tissue extracts, and when used in conjunction with immunoblotting and specific antibodies to proteoglycan core protein and glycosaminoglycan, side chain epitopes can provide significant information on the level of proteoglycan polydispersity/heterogeneity and a number of proteoglycan populations present in tissue samples. This can be a technically difficult technique, but it reveals significant information on proteoglycans from small tissue samples not possible by any other separation methodology. Native full-length and proteoglycan fragments are examined in this technique something which cannot be done in the popular SDS-PAGE format unless the glycosaminoglycan side chains are first removed. Furthermore, since proteoglycans do not require renaturation from SDS-protein complexes, the proteoglycan populations separated by native electrophoresis are highly reactive with antibodies in immunoblotting procedures. Despite the massive sizes of proteoglycans, transfer conditions have been determined which provide close to quantitative transfer to nitrocellulose membranes without exceeding the binding capacity of such membranes, avoiding bleed-through of the transferred proteoglycans. Development of biotinylated hyaluronan and its application in an affinity blotting procedure has also yielded significant information on aggregatable proteoglycan populations separated by CAPAGE from a number of cartilages and vascular tissues in health and disease. While the CAPAGE system can be a technically demanding technique to master particularly in gel preparation, all other steps are straightforward, and the method yields invaluable information on proteoglycan populations extracted from connective tissues in health and disease that cannot be ascertained by any other technique. Further improvements in the detection of proteoglycan features with the development of novel bio-affinity probes or new antibody preparations are expected to further improve the utility of CAPAGE separation methodology.