Challenge question .The image below shows the results of two Western Blots after
ID: 204946 • Letter: C
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Challenge question.The image below shows the results of two Western Blots after performing SDS-PAGE. In Gel A , a polyclonal 1 antibody against phosphorylated Thr 203 and Glu 204 in MAPK was used. in Gel B, an anti-total 1 MAPK polyclonal antibody that binds both the phosphorylated and dephosphorylated forms was used. This anti-total MAPK antibody was produced from a small region of of the MAPK polypeptide, but this fragment is from a region away from Thr 203 and Glu 204. In both filters, a 2 monoconal antibody was then used to bind to the constant region of the anti-MAPK 1 antibody and produced a signal that allowed to visualize the MAPK bands on both Western blots at 42 kDA and 44 kDA. On top of each Western Blot, duplicates of various concentrations of fibroblast growth factor 1 (FGF-1) were added, including a negative control (0 ng/ml FGF-1). Interpet these results by determining which concentration(s) was or were most effective at increasing MAPK activity. Consider the change in intensity of the bands from Gel A to Gel B, as well as betwen the bands in each condition duplicate. How can the loading and negative controls raise doubts about the validity of the experiment?
Gel A anti-phospho 42kDa 44kDa Loading control Gel BZ anti-total 42kDa 44kDa Loading controlExplanation / Answer
As we can see in the first gel that the level of phosphorylation of MAPK when the cells were treated with 1ng/ml and 10ng/ml FGF-1, levels of phosphorylation is more in 10ng/ml treated sample. But the loading control in case of a 10ng treated sample is also increase. So, 1ng/ml FGF-1 level is more effective in phosphorylating MAPK.
In case of 0.1ng/ml treated sample, the sample which is near to the ladder gas more protein as compared to the sample in the lane 3 which shows that the loading was not equal.
In case of negative control, ideally there should not be any phosphorylation but as we can see in the gel picture that MAPK is phosphorylated. Phosphorylation in negative control shows that there are some factors which are phosphorylation MAPK at Thr 203 and Glu 204.
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