This is a BIochem Lab Question Help Please 2 You are given t

This is a BIochem Lab Question. Help Please!

2. You are given the following SDS-PAGE gel to interpret. Lane 1 contains molecular weight markers. The gel on the right (from the manufacturers website) is of the same standard MWM mixture, and provides the molecular weights for each protein. 1 2 3 Protein A: p 9 Protein B: pl 6 Protein C: pl 4 Protein D: pl 9.5 Protein E: pl -3 a) Do you think Coomassie Blue or Western Blot sta ning was used to visualize this gel? Briefly, explain your answer. b) Why might the molecular weight markers migrate differently (ie: have different spacing) in the two different gels? (hint: how do SDS-PAGE gels separate proteins?) c) Which protein is smaller, A or E? Briefly, explain your answer. d) Approximately what moleculear weight is protein B? (hint: look at the intensities of the bands in the MWM, count bands up from the bottom, and match between left and right). 3. You have the mixture of proteins that was loaded in lane 2 of the gel in question 2. Using the information provided above, design a good column chromatography procedure using the tools learned in class that will separate out protein B from the mixture. You will need to describe the column that you selected, any buffers required, and any steps required to recover the protein.

Solution

A) for the staining comaasive blue was used that blue colour on the gel is indicating that its looking like a gel not blot for western we need to transfer this in nitocellulose or PVDF membrane followed by probing with specific antibody.

B) Running can be affected with different parameter like percentage of acrylamide, salt in the sample, PH of tris and running buffer. but as mentioned there is spacing difference menas acrylamide perecntage of gel is different.

C) E is smaller because this protein running fast in SDS-PAGE and lower moleculer weight protein run fast in the SDS-PAGE

D) it is just above the 4 band of marker protein from bottom of the gel and in marker lane this band coorespond to 30 kDa so protein B having molecular weight little bit higher than 30. (please consider that the marker lane is not clearly visible)