Question

You suspect that your purified E protein binds a segment of host (human) DNA required for the expression of a host protein essential for the mammalian immune response.

A. How would you show that your purified E protein binds specifically to this segment of DNA?

B. What controls would you use to ensure that the binding is specific?

C. How would you determine the apparent Kd of the purified E protein for this DNA?

Answer 1

Solution:

A. The specific sequence where purified E protein binds can be altered. There must be specific sequence on the DNA sometimes specific consensus sequence. Alteration to this sequence also alters the binding site of E protein. Thus by altering the sequence and analysing the binding of E protein,and downstream regulations we could conclude that the E protein always binnds to a specific site or element on the DNA.

B. To analyse the binding is specific we divide the experiment in to three groups.

Group 1 : Segment of the DNA that does not possess the binding element serve as negative control.

Group 2 : Segment of DNA with mutation on elements where protein binds (Test group)

Group 3 : Segmnt of the DNA with binding sites present (positive control)