TECHNIQUE FOR RESOLVING COMPLEX MIXTURES OF PROTEINS.

TECHNIQUE FOR RESOLVING COMPLEX MIXTURES OF PROTEINS. HOWEVER, THE METHOD HAS HAD LITTLE CLINICAL USE BECAUSE IT IS NOT QUANTITATIVE. THE WORK PROPOSED HERE IS DIRECTED TOWARD CAREFUL QUANTITATION, USING 2-D ELECTROPHORESIS, OF 6 TEST SERUM PROTEINS WHICH CAN BE PURCHASED AND ARE WELL RESOLVED ON A 2-D GEL. THE PROTEINS ALBUMIN, TRANSFERRIN, ALPHA-ANTITRYPSIN, APO-AL LIPOPROTEIN, IGG HEAVY CHAIN AND HAPTOGLOBULIN B CHAIN MEET THESE CRITERIA. THEY WILL BE PURIFIED BY IMMUNOPRECIPITATION FOLLOWED BY PREPARATIVE 1- OR 2-D ELECTROPHORESIS. THE PURIFIED STANDARDS WILL BE RADIOLABELED BY REDUCTIVE ALKYLATION WITH 14C-LABELED FORMALDEHYDE SO THAT RECOVERIES IN THE FINAL SLAB GEL CAN BE MEASURED BY COMPUTERIZED QUANTITATIVE AUTORADIOGRAPHY. STANDARD CURVES OF STAIN DENSITY VERSUS G PROTEIN WILL THEN BE CONSTRUCTED FOR EACH PURIFIED PROTEIN. THE STANDARD CURVES ALONG WITH PROTEIN RECOVERY DATA WILL BE USED TO CALCULATE CONCENTRATIONS IN UNKNOWN SERUM BY COMPUTERIZED DENSITOMETRIC ANALYSIS OF STAINED 2-D GELS USING THE MCIDAS SYSTEM AT THE UNIVERSITY OF WISCONSIN. THIS DATA WILL BE USED TO FINE-TUNE AN INEXPENSIVE TELEVISION CAMERA-MICROCOMPUTER SYSTEM FOR ROUTINE ANALYSES. IF SUCCESSFUL, THIS PROJECT WILL SET A PRECEDENT FOR COMMERCIALIZED QUANTITATION OF CLINICAL SAMPLES USING 2-D ELECTROPHORESIS.