WESTERN BLOT MARKER WITH UNIVERSAL IMMUNOGLOBULIN BINDING DOMAIN

Open Access
Author:
Fleischman, Andrew
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Song Tan, Thesis Supervisor
  • Ming Tien, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • western blot marker
  • IgG Binding Domain
  • western blot
  • protein a
  • molecular weight marker
  • immunblot
Abstract:
Immunological techniques such as western blotting harness the specificity and avidity of antibodies to detect proteins. In western blotting, antibodies specifically detect proteins separated by SDS-PAGE electrophoresis. Molecular weight markers are important components of a western blot because they allow one to verify protein sizes. However, proteins in a traditional molecular weight marker will not appear on the film of a western blot, since the various primary antibodies cannot recognize them. The goal of this thesis research is to develop universal western blot molecular weight markers that bind commonly used secondary antibodies. The IgG Binding Domain of Protein A (PRA), a domain that binds the heavy chain region of antibodies, was fused to proteins of various sizes. Each recombinant construct was expressed and purified on a small scale to determine the optimal expression conditions and to check the solubility of the protein. These optimal conditions were then used to express the protein on a larger scale. Each marker protein was purified by metal affinity chromatography via the N-terminal polyhistidine tag. The purified proteins were combined into a mixture, producing a set of western blotting molecular weight markers that be used independent of the primary antibody employed.