Week 9

I previously talked about how I expressed the TEV G>S cleavage site in bacterial cells. Not much happened last week, I was mostly focusing on working on my presentation. I scanned the SDS Page gel image I had that confirmed the TEV cleavage site was successfully expressed as protein. Then my mentor had to purify the protein because he said it was very complicated and needed everything to work well the first time. This week and next week I am running Bradford assays with the purified protein to determine the concentration of protein I have. The Bradford assay involves the use of Coomassie Brilliant Blue dye binding to specific protein of interest in order to determine the absorbance of that protein. Coomassie Brilliant Blue dye has three variant forms: red under acidic conditions, blue under basic conditions, and green under neutral conditions. When the dye binds to the protein of interest, it changes color from red to blue. The darker blue the dye is, the higher concentration of protein is bound to the dye. A spectrophotometer shines UV light through sample and detects the blue protein dye form at 595 nanometers of wavelength. Specific volumes of Coomassie Brilliant Blue dye will be added to known concentrations of the protein Bovine Serum Albumin and the absorbance of each variant solution will be detected with a spectrophotometer. A standard curve of absorbance vs. concentration will be generated with this to determine the unknown concentration of the protein I am testing.