Author

Aunaly Palmer

Publication Date

2013

Document Type

Honors Thesis

Department

Biochemistry

Keywords

Proteomics, Mice-Muscles, Myogenesis, Proteins-Analysis, Desmin regulation, Murine C2C12 cells

Abstract

Desmin is a type III intermediate filament protein found in the cytoskeleton of skeletal muscle. Mutations in the gene encoding for desmin has been shown to cause early onset cardio myopathy and extreme muscle weakness. The expression of desmin and its various post translational modifications were observed during myogenesis. Murine C2C12 cells were harvested at the myoblast, the early myotube, and the late myotube stage. The total protein at each of the time points was isolated and visualized using two-dimensional SDS gel electrophoresis stained with Coomassie brilliant blue R-250. The first dimension separated the proteins based on overall charge, isolating only those with isoelectric points between pH 4.7 and 5.9. Two-dimensional immunoblots of the three stages were produced using two different clones of anti-desmin antibodies. The DE-U-10 anti-desmin monoclonal antibody was made using the full desmin protein, whereas the B-7 anti-desmin monoclonal antibody was made using only amino acids 413-445 of desmin. The DE-U-10 immunoblots showed binding to a larger variety of protein species than the B-7 antibody. Observation of the immunoblots suggests that the amount of desmin in the cell increases during myogenesis, especially during the transition from myoblast to early myotube. The images of the immunoblots and the gels were overlaid using PDQuest Advanced 2D Analysis Software v8.0.1. The spots identified as possibly contain desmin due to the overlap in the immunoblot and gel were manually excised. Spots thought to contain actin were also excised. The protein species within the spot were digested with trypsin and analyzed by high pressure liquid chromatography mass spectrometry. Of the 28 spots analyzed, 16 were found to contain desmin, four were found to contain vimentin, and eight were found to contain both desmin and vimentin.

Language

English

Comments

v, 75 pages : illustrations (some color). Honors Project-Smith College, 2014. Includes bibliographical references (pages 72-75)

Share

COinS