Question

*Biochemistry Lab Question

1. Chromatography columns have a limited protein binding capacity (Consider not overloading the column) Yet you may have noticed that the Wash fraction fluoresced slightly. If you see a band in the Western Blot Wash lane, what does this data say about the physical protein structure of rGFP in that lane? Give a possible MW for this band.

2. (4pts) If overloaded the Ni⁺² Agarose column with a crude extract sample that contained too much rGFP, would you expect to see a band in the Wash lane of the Western Blot? If so what is its MW? Would you expect to see a band in the E2 lane? If so what is its MW?

** I know the washes could not have contained and histidine bound rGFP but loosing the n-term would also mean loosing the express tag right? in this case I thought it would not be detected on western blot. Please explain answers :)

lab technique question / Not research

Answer 1

Answer:

1.In the event that the protein is seen in W2 path, it proposes that protein has not bound to the Ni+2 segment and physical nature of protein might be the histidine destinations may have been covered inside the protein, which makes the protein unfit to tie. The MW of the protein may same as that of the local protein.

2. Less measure of protein would be found in the W2 path because of the over-burdening of the example. In the E2 path, more measure of protein can be viewed as all the protein bound to the segment will be eluted. The MW of protein in W2 and E2 path would be same as the two proteins are same.