Question

Betergents h. What chemical would you add to refold this protein? 16. You wish to purify a novel protein from mammalian cells. You do not know this protein's size or charge, and are thus unsure of which steps to take. Fortunately, an antibody for this protein is available. You have two cell lines: one that expresses the protein, and one • that does not. . ) 4 5 III111 You run both cell lines on an SDS-PAGE (A) followed by a western blot using the same gel(B). Lane 1 shows the control cell line, and Lane 2 the expression cell line. The cells that express the protein were homogenized, and the soluble (Lane 3) and insoluble (Lane 4) fractions are shown. a. Which lane (3 or 4) contains the protein of interest? b. Is it soluble or insoluble? C. Two new proteins are visible in this fraction. Which (the top or bottom band) is the target protein? How are you certain? d. What is the molecular weight of the protein of interest? e. What is the molecular weight of the expressed contaminant? The fraction from Lane 4 was placed on a cation exchange column and the flowthrough (Lane 5) and elution (Lane 6) fractions are shown. f. Which would you keep from IEX, the flowthrough or elution? g. At the pH used, what is the charge on the proteins in Lane 5? Lane 6?

Answer 1

16.

a. Lane 4 contains the protein of interest.

b. The protein is insoluble because it has been detected in lane 4 which is insoluble fraction.

c. The protein visible on western blot is around 20kDa. So the target protein is the protein which is showing molecular weight around 20kDa.

d. Molecular weight of protein of interest is around 20kDa.

e. I can see there are two contaminants in the fraction from lane 4. The upper contaminant is around 36-38kDa and lower is around 10 kDa.

f. We have to keep elution since it contains protein of interest.

g. Cation exchange column is used to purify positively charged protein.

So, charge on protein in Lane 5- negative and in Lane 6- positive