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Using affinity purification -- mass spectrometry to identify aryl hydrocarbon receptor nuclear translocator interacting proteins

Author

Ashraf G. Madian, University of the Pacific

Date of Award

2005

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Chemistry

First Advisor

Jianhua Ren

First Committee Member

William K. Chan

Second Committee Member

Patrick Jones

Abstract

The aryl hydrocarbon nuclear translocator (ARNT) belongs to the family of basic helix loop helix proteins. ARNT forms a heterodimer with aryl hydrocarbon receptor (AhR). This heterodimer binds to the dioxin responsive element (DRE) causing the regulation of the gene expression of some enzymes such as CYP1Al. Studies show that the ARNT-AhR heterodimer needs protein factors to bind to DRE, and most of these protein factors are still unknown. ARNT also heterodimerizes with hypoxia inducible factor 1 a (HIF-1 a) which mediates the cellular responses to hypoxia. The purpose of this research is to use a combination of affinity chromatography and mass spectrometry techniques to identify the proteins that interact with ARNT.

We chose mouse liver as the protein source. We planned to isolate the ARNT interacting proteins from the mouse liver protein extract by using the TALON® resin

column bounded with 6x HIS-ARNT. Two negative control experiments were done. The first one by the application of the liver extract only on non-ARNT bound column. The second one by the application of 100 mg of bovine serum albumin on 6x HIS-ARNT bound column. The mouse liver extract was applied on 6x HIS-ARNT bound column. The column was washed with an increasing concentration of potassium chloride (0.05 M- 1 M). ARNT was eluted with a buffer containing 250 and 500 mM imidazole. The different washing fractions were compared with the negative control experiments. There was no difference between this and experiment and negative controls. We also tested using in-vitro chemical cross-linking with formaldehyde. Some distorted bands that may '· represent crosslinked proteins appeared above ARNT molecular weight by the addition of 1% paraformaldehyde for 20 minutes at 37°C, and for 2 hours at 30° and room temperatures. These bands were absent in the negative control experiments. The mass spectrometric protocols for identification of trace amount of proteins using peptide mass fingerprinting were tested using a standard protein (Bovine Serum Albumin).

Pages

106

Recommended Citation

Madian, Ashraf G.. (2005). Using affinity purification -- mass spectrometry to identify aryl hydrocarbon receptor nuclear translocator interacting proteins. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/605