Question

Some characteristics of three proteins are listed in the table below:

Protein	Molecular Weight (Da)	Isoelectric point (pI)	Does the Protein Contain a heme moiety?
1	65,000	5.5	No
2	54,000	10.0	No
3	14,500	4.5	Yes

a.(2 points) What type of chromatography separates proteins based on size?

b. (2 points) What type of chromatography separates proteins based on their charge?

c.(10 points) Could gel filtration chromatography be used to separate a mixture containing Protein 2 and 3? Clearly explain why or why not. If gel filtration chromatography can be used to separate Protein 2 from Protein 3, which protein would elute first (clearly explain why)?   After collecting the fractions from the column, the absorbance of each fraction will be measured using a spectrophotometer. Can both proteins (Protein 2 and Protein 3) be monitored at 280nm and 400nm (clearly explain)?

d. (6 points) Which 2 proteins listed in the table above could be separated by ion exchange chromatography but not by gel filtration chromatography (clearly explain why)?

Answer 1

(a) Size exclusion chromatography is used to separate proteins based on their size.

(b) Ion exchange chromatography separates proteins based on their charge.

(c) Gel filtration chromatography will be able to separate protein 2 and 3 as there is a large difference between their Molecular weight. Protein 2 is larger in size compared to protein 3. The protein with a larger size (protein2) will be eluted first as it is too large to fit into any pores. Two proteins can not be monitored at 280nm and 400 nm as one contains heme group but other one does not.

(d) 1 and 2 can be separated by ion exchange chromatography due to difference in their charge. However they have similar size and can not be separated by gel filtration chromatography