Isolation of potential protein targets of MS-818 using affinity chromatography
According to the National Institute of Neurological Disorders and Stroke, there are more than 600 neurologic disorders that affect approximately 50 million Americans each year. The $91 billion dollars spent by Medicare on Alzheimer's disease and other dementias in 2005 is projected to increase to $189 billion by 2015 . The existence of neural stem cells (NSC's) and neurogenesis makes neural regeneration a viable option. The ethical barriers of using embryonic stem cells, rejection of the transplanted cells, and possible tumor formation, are only a few of the problems that face stem cell transplantation, a widely considered option to repopulate the brain with cells. A noninvasive pharmaceutical approach that can promote neuron regeneration and recovery would be the key to curing many neurodegenerative diseases. The development of MS-818 as a non-invasive enhancer of the proliferation process of NS Cs is revolutionary for the treatment of neurodegenerative diseases. Due to the fact that its mechanism of action remains unknown, the full pharmacological potential of MS- 818 has not been fully exploited . Isolating protein targets of MS-818 is key in identifying the molecular pathways responsible for its mechanism of action. UV-Vis analysis of MS-818 showed absorbance at 275-nm, and this data was used to calculate coupling yield. MS-818 coupled to the NHS-activated sepharose beads of the affinity column with 83% efficiency. Proteins were isolated from human embryonic kidney cells (HEK 293 cells) and applied to the column. Bradford assay confirmed that bound proteins eluted in a concentration dependent manner when an MS-818 gradient was applied to the column. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the eluate revealed two sets of polypeptides migrating at 37-75 kDa and 100-150 kDa. In addition, some trace polypeptides in the sub-35 kDa region could be seen. Although we have not yet identified specific proteins that MS-818 interacts with, we were able to successfully to isolate such proteins.
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Bachelor of Science (B.S.)
College of Medicine
Molecular Biology and Microbiology
Dissertations, Academic -- Medicine;Medicine -- Dissertations, Academic
Length of Campus-only Access
Honors in the Major Thesis
Jaffal, Jad M., "Isolation of potential protein targets of MS-818 using affinity chromatography" (2010). HIM 1990-2015. 1010.