2. A research group is studying how a new drug compound affects melanoma (skin c
ID: 253842 • Letter: 2
Question
2. A research group is studying how a new drug compound affects melanoma (skin cancer) cells. They grow the cells in the wells of a culture dish (pictured on the right). To different wells they add drug compound at the following concentrations: 0 ??. 62.5 ??, 125 ??, and 250 ??. After 24 hours they harvest the cells from each well and make a lysate. They run samples of the lysates on SDS-PAGE gels for analysi:s A Cell Supernatant: Contains Protein Supernatant removed and kept. Junk Pellet ../ Centrifugation drug In panel A is an image of a gel that they stained directly with Coomassie (Note: Coomassie is a dye that binds aromatic side-chains in proteins). In panel B are portions of five western blots performed with antibodies to the following proteins: STAT3, Cdk4 cyclin-D1, cyclin-D2 and beta-actin. Each lane is labeled with the drug concentration with which the cells used to make the lysate were treated. [kDal 0 62.5 125 250 HM drug 0 62.5 125 250 25* STAT-3 150. 102. 52. Cyclin-D2 a. What can you say about the amounts of all proteins 3s. in the different samples? Explain how the data support31» your statement. ?-Actin 24. b. Does the drug have an effect on the overall protein levels in these cells? c. What can you say about the amounts of beta-actin in the different samples.? Explain how the data support your statement. d. beta-actin is known as a house-keeping gene because its protein levels vary little between samples Explain how beta-actin can therefore be used as a "loading control" to be sure the same amount of lysate is loaded in each gel lane. Why is that important? e. What can you say about the amounts of STAT3, Cdk4, cyclin-D1, cyclin-D2 and beta-actin in the different samples? Explain how the data support your statement. f. The expression of STAT3, Cdk4, cyclin-D1 and cyclin-D2 are known to promote cell division. What effect do you predict the drug will have on cell division?Explanation / Answer
Answers
A. As per the gel image (A), there is no difference seen in amount of any protein, but if we see the image (B), then we can say that STAT-3 and Cyclin D1 are gradually decreased with increse in drug concentration. where beta ctin and cdk4 are unchanged in all samples. Cyclin D2 is not present in any of the samples.
B. as per the image A, drug dont have overall effects on the protein level of cell, it has just effect on targeted proteins only. so in image, it seen that, only two proteins are affected with its concentration.
C. Image B, shows that beta actin doesn't has any effect of this drug, its shows same intensity band in each and every sample.
D. As per the information given in question, it is good thing that beta actin like housekeeping gene is not affected with this drug, and it is showing contineous intensity band in all the samples, which proves that the amount of lysate is loaded equally in each and every well, so it is serving as a loading control and also as a negative control. if lysate loading changes in different wells, then our data does not have any perticular meaning of comparision, because band intensity is based on amount of protein present in loading volume, and so if loading amount is more means more intensity. so constant and fixed volume should be loaded.
E. STAT -3 is gradually decreasing in all increasing concentration. where Beta actin and cdk4 are not changing in any of the sample. so this drug dont has any effect on these proteins. Cylcin D1 showing very good effects of this drug. it is affects more compare to STAT -3. where cyclin D2 is not showing any band even in 1st sample (0 micro gram).
F. if these all proteins are required for promote to cell division then cell division will be affected potentially. but if some of them or anyone of them is required then cell division has not that much affected this drug.
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