Electrophoretic Characterizations of Cross-Linked Fibrinogen Derivatives in Blood and Vascular Tissue by Zonal Immobilization on Glyoxyl Agarose
Current definitions of fibrinogen derivatives produced in course of clot formation and lysis derive from electrophoretic and immunochemical analyses of the alterations that the purified protein undergoes during these reactions (1, 2). Methods for profiling the distribution of fibrinogen-derivatives in blood continue to improve as new methods arise for immunoprobing electrophoregrams. Polyacrylamide gel electrophoregrams (PAGE) are too impermeable to be probed directly with antibody. The earliest profiles of molecular weight distributions of the fibrinogen-related antigens in blood used immunosorbents for specific absorption of the protein from blood, followed by electrophoretic analysis of desorbed protein (3). Western-blotting (4) in which proteins are electro-transferred out of the electrophoregram to produce a replica on cellulose nitrate or other adsorptive membrane is now commonly used for immunoprobing. However, as might be anticipated (5), this technique does not work well with fibrinogen; it and its polymers do not transfer well due to their precipitation when separated from SDS. Because of relatively poor transfer of the intact protein, blots made of fibrinogen-related antigens usually show the degraded and readily transferred forms at levels far out of proportion to their actual concentrations in the samples. To eliminate the uncertainties that arise from transfer procedures, we have devised methods for directly immunoprobing the electrophoregrams. The present communication outlines underlying principles of the methodology, and surveys some of the findings regarding the composition of fibrinogen which were discovered with it.
KeywordsAortic Intima Zonal Immobilization Early Profile Glyoxyl Agarose Neutral Phosphate Buffer
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