You need to separate a mixture of proteins of expected sizes: 12 kDa, 45 kDa, 82

Question

You need to separate a mixture of proteins of expected sizes: 12 kDa, 45 kDa, 82 kDa, and 160 kDa using an appropriate gel filtration column. The lab has the following choices:

(A) Sephadex G-50 (fractionation range 2000 – 30000)

(B) Sephadex S-75 (fractionation range 3000 – 70000)

(C) Sephacryl S-200 (fractionation range 5000 – 250000)

(D) Sephacryl S-400 (fractionation range 20000 – 800000)

a. Which column material would you use to achieve the best separation of the listed protein sizes?

b. A 75 ml column was packed using the right material. What elution order will you expect for your proteins assuming all the proteins are behaving as monomers in the particular buffer you chose for the experiment (i.e., which comes out first, second, third and fourth, write the molecular weight of the proteins in the order of elution)?

c. The protein mixture was purified in a different buffer than in section 5b. The resulting chromatogram and the molecular weight corresponding to each of the peak based on molecular weight standards are shown below. Based on the principles of gel filtration chromatography, how can you explain the chromatogram? (Hint: use the molecular weight and compare the oligomerization state of the different components in the mixture to identify what each peak represents)

Explanation / Answer

Gel filtration chromatography seperation is based on the size of the molecule. Its basically size-exclusion chromatography where the eluent used is an aqeous solution and thus known as gel filtration chromatography. Here the matrix used is a porous material. It is stabilised with a buffer to which an eluent is added that has particular pH, necessary ions,protease inhibitors etc. As the sample is injected, the smaller particles are found to interact with the matrix more than that of larger sized ones. Thus as the eluent is passed larger molecules comes out first and the smaller the last.

a) The column (C) is preferable as the range of proteins to be seperated lies in the range 12000 - 160000 Da. This column has a range of 5000 - 250000 Da. In case of A, B there are chances that it will come as mixture, not properly seperated. In case of D it will take more time for elution.

b) The elution will be as mentioned above, the larger ones elute first follwed by the smaller ones.

The decreasing order of elution would be

160 KDa > 82KDa > 45KDa >12KDa

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