Access Type

Open Access Thesis

Date of Award

January 2013

Degree Type

Thesis

Degree Name

M.S.

Department

Biochemistry and Molecular Biology

First Advisor

Robert Akins

Abstract

Abstract

LC3I and LC3II as Autphagy Markers for the Development and Improvement of Products and Techniques used in Research

By

Caitlin J. Williams

May 2013

Advisor: Dr. Robert Akins

Major: Biochemistry & Molecular Biology

Degree: Master of Science

Autophagy is an intracellular process that functions to maintain homeostasis in the cell by degrading misfolded proteins, old or nonfunctioning organelles, and outside invaders such as bacteria or viruses. This process can be split into three different types, microautophagy, chaperone mediated autophagy and macroautophagy. Macroautophagy is the most commonly studied form and is believed to be regulated by Atg proteins, as well as cargo proteins that bring debris to the autophagosome. Macroautophagy is characterized by 5 steps including initiation, elongation, maturation, autophagosome-lysosome fusion and lysosome degradation. Autophagy has been found to be involved in diseases, such as cancer and neurological disease. LC3I and LC3II function in late stage autophagy before lysosome fusion. The study of a complex process such as autophagy requires the development of tools to enable precise and quantitative research. The ability to produce antibodies to key autophagy proteins, methods of cellular fractionation and fractionated control cell lysates provides product development opportunities for Enzo Life sciences and new research tools for researchers. To support these needs, methods of cell fractionation were developed along with antibodies specific to LC3I and LC3II. Cell fractionation methods were optimized to enable separation of LC3I and LC3II. The optimized methods were shown to be effective on multiple cell lines with the use of western blotting. Due to the difficulty in achieving stable hybridomas monoclonal

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antibodies that bind both LC3I and LC3II haven't been identified. The LC3II antibody development failed to produce antibodies in western blotting. Efforts are ongoing to identify stable hybridomas producing antibodies to LC3. The antibodies should recognize both LC3I and II. Combining the use of these antibodies with fractionated cell lysate methodology should allow quantitative detection of LC3II by ELISA. Such a tool will be an important tool for autophagy researchers.

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