Designing Optimized Protein Molecular Weight Markers Applicable to both Coomassie Staining and Western Blotting
Open Access
Author:
Shibata, Yoshitaka Joey
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
Song Tan, Thesis Supervisor Wendy Hanna-Rose, Thesis Honors Advisor
Keywords:
protein molecular weight markers SDS-PAGE Western blotting IgG antibody binding polycistronic expression protein expression protein purification
Abstract:
Molecular weight markers are essential tools to measure protein sizes by gel electrophoresis. However, conventional markers are not detected in Western blots which use antibodies to recognize specific proteins. Together with undergraduate colleagues, I have designed and prepared 11 recombinant proteins of defined molecular weights from 10 to 250 kDa that contain Staphylococcus aureus Protein A IgG antibody binding domains through subcloning. These recombinant proteins can be visualized on polyacrylamide gels with Coomassie Blue stain and also on Western blots with non-specific secondary antibodies. In addition to migrating appropriately on SDS-PAGE gel, each recombinant protein was selected for high level expression in E. coli and contains a HIS tag for efficient metal affinity purification. In addition, two set of polycistronic expression vectors were created to simplify the expression and purification processes. The first vector co-expresses 10, 30, 50, and 100 kDa proteins, and the second vector co-expresses 20, 40, 60, and 80 kDa proteins at 21C. The Penn State protein molecular weight markers should provide an inexpensive method to produce protein molecular weight markers for research laboratories.
