You decide to study three proteins--Protein A, Protein B, Protein C--with potent

Question

You decide to study three proteins--Protein A, Protein B, Protein C--with potential roles in transcriptional regulation in mammalian cells. You perform an electrophoretic mobility shift assay (EMSA). The data is shown below. All reactions contained binding buffer and labeled DNA fragment that included the sequence for the DNA-binding site for Protein A, a sequence upstream of a mammalian gene. The added purified protein(s) are indicated above the gel.

Based on this data, does Protein B alone bind DNA? Explain your answer.

a) Yes, this can be concluded from the comparison of Lane 2 and Lane 1.

b) No, this can be concluded from the comparison of Lane 2 and Lane 1.

c) No, this can be concluded from the comparison of Lane 5 and Lane 1.

d) Yes, this can be concluded from the comparison of Lane 5 and Lane 1.

What about protein A? Protein C?

Explanation / Answer

B) No, this can be concluded from the comparison of Lane 2 and Lane 1
In the column 5, there are three bands that are being observed. Lane 1 is negative control and contains only genetic material. When the DNA would be bound with any of the proteins there would be a single band that would be half the width of the genetic material. Here , in lane 4, there are 2 bands in the presence of protein A . This implies that Protein A alone is binding the DNA. If protein B was also binding the DNA, the band appearing in Lane 2 , with only protein B would be similar to this band rather than being same as the negative control. Since, Lane 2 is the exact replica of Lane 1, that means presence of protein B is similar to presence of no protein at all, since it does not have a binding effect. Thus, Protein B alone does not bind the DNA.

Protein C is similar in nature to Protein B , i.e. it does not have any effect on the binding of DNA. The only binding of DNA is done by protein A.This also goes with the fact that the binding buffer contained labeled DNA fragment that included the sequence for the DNA-binding site for Protein A.

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