Date of Defense

8-27-1993

Department

Biological Sciences

First Advisor

Leonard Ginsberg, Biological Sciences

Second Advisor

Gus Walker, Upjohn Company

Third Advisor

Larry Gross, Upjohn Company

Abstract

CD4-PE40 is a recombinant protein that contains variations in its primary sequence which give rise to charge isoforms. Because it is a potential protein pharmaceutical, methods are needed in order to detect and characterize these variants. Analytical separation of the isoforms was done using polyacrylamide gels with a pH range of 4-6.5. Initially, recovery of protein from the gel was done using electroelution and a 0.3M 1.5:1.0 Tris-CHES elution buffer. Electroeluted samples were then digested with trypsin to detect differences between the bands as evidenced by differences in each peptide map. Because of low protein recovery using electroelution and difficulty reproducing the tryptic maps, it was not pursued further. As an alternative, electroblotting using PVDF membranes because the focus for protein recovery. Although recovery was improved, reproducibility was still questionable. Before conclusions can be drawn regarding solid differences between the bands, the procedures for digestion of protein in situ and subsequent tryptic mapping of the peptides must be optimized first.

Access Setting

Honors Thesis-Campus Only

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