A rapid one-step extraction procedure for the isolation of ubiquitin from human erythrocytes for antibody production.
A procedure is described that employs 5% perchloric acid extraction to isolate ubiquitin from human erythrocytes. The procedure is rapid and economical as it requires no specialized equipment. The extracted protein appeared to be highly purified as judged by electrophoresis and was identified as ubiquitin by immunoblotting and total amino acid analysis. The extraction yields about 78% of the ubiquitin in the hemolysate, which is a higher yield than is obtained with other procedures. The purified ubiquitin was used to make a polyclonal antiserum. As ubiquitin is a small and highly conserved protein, it is necessary to couple it to a larger immunogen to elicit an immune response. This ubiquitin antiserum was produced using an immunogen system that produces an immune response to the ubiquitin, but not to the carrier protein.
Sparkman
D R
DR
Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235-9072.
Hill
S J
SJ
White
C L
CL
D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000737:chemistry*; D006461:Hemolysis; D006801:Humans; D010472:Perchlorates; D014452:Ubiquitins / Q000276:immunology / Q000302:isolation & purification*
D010472:Perchlorates; D014452:Ubiquitins