Assay of protein turnover using a bioluminescent reporter

Assay of protein turnover using a bioluminescent reporter Affiliation Results and Discussion Answer to Question The results were relatively different for the corresponding LUCWTs and LUCPs. For the sample LUCWT, light emitted in 10 seconds per ul (RLU/ul) at time zero was 1236. 8 RLU/ul. The average protein concentration at time zero was 0. 53 ug/ul while the Relative Luciferase Activity at time zero was 2333. 58. For the sample LUCP, light emitted in 10 seconds per ul (RLU/ul) at time zero was 1205. 5 RLU/ul. The average protein concentration at time zero was 0. 53 ug/ul while the Relative Luciferase Activity at time zero was 2274. 47. The difference could have been caused by various factors including pipetting errors (Gould & Subramani, 1988).
2. Answer to Question 2
An estimate of a half-life for the LUCP enzyme is (How to Calculate Half Life, 2015):
Half-life = t*In 2
10seconds = t*0. 301
t= 10seconds*0. 301 = 30. 1 seconds.
3. Answer to Question 3
It is important for some proteins to have a short half-life but no others like clusterin because they have to control excessive growth of cells. Clusterin in this case controls prostate cancer cells (Rizzi, Caccamo, Belloni, & Bettuzzi, 2009).
4. Answer to Question 4
In designing an experiment using MetLUC to assay the efficacy of three compounds with respect to BF A, the first thing to consider is that the activity of BF A is known. The BF A would thus be used as the control variable (Schultz, Cegielski, & Hastings, 2005). Analysis of each of the three novel compounds would be done. This would be based on their respective signal sequence, in which each of them would be expressed in tissue culture cess. Comparison of how the MetLUC protein for each compound would be translocated to the ER and the resulting secretion into the medium in which the cells are growing would be done. The results of each would be compared to the known facts about the same experiment in BF A.
References
Gould, S., & Subramani, S. (1988). Firefly luciferase as a tool in molecular and cell biology”. . Anal. Biochem. 175 (1), 5–13.
How to Calculate Half Life. (2015). Retrieved from wikihow: http://www. wikihow. com/Calculate-Half-Life
Rizzi, F., Caccamo, A., Belloni, L., & Bettuzzi, S. (2009). Clusterin is a short half-life, poly-ubiquitinated protein, which controls the fate of prostate cancer cells. Journal of Cell Physioly, 19(2), 14-23.
Schultz, L. L., Cegielski, M., & Hastings, J. (2005). Crystal structure of a pH-regulated luciferase catalyzing the bioluminescent oxidation of an open tetrapyrrole . Proc. Natl. Acad. Sci. U. S. A., 102 (5), 1378–83.