M.W. pI Proteoform Phosphorylation Hydrophobicity Protein A 50 kDa 10 0, 1, 2 -0

Question

                  M.W.             pI             Proteoform Phosphorylation         Hydrophobicity

Protein A   50 kDa           10             0, 1, 2                                          -0.25

Protein B   80 kDa            6               0                                                   -0.5

Protein C   10 kDa            3               0                                                   -0.14

1. Please draw schematic figures for the results of the following approaches using the protein mixture in the table above: (1) RPLC; (2) anion exchange column (buffer pH =8); (3)SEC; (4) cation exchange column (buffer pH=8)

2. Which type of 2D HPLC separation combination can potentially resolve all the proteoformsin the protein mixture? Can you illustrate and explain it step-by-step?

Explanation / Answer

1. SEC(Size exclusion chromatography)-In this kind of chromatography, molecules separate through their size where larger molecules elute from the column faster and smaller molecules later. Thus result/Elution will be Protein B-Protein A-Protein C.

anion exchange column (buffer pH =8)- When pH of the protein of interest is greater than the pI then the protein will carry a net negative charge, thus result will be Protein A will elute in an unabsorbed fraction means during washing and in eluted fraction Protein C then Protein B.

Cation exchange chromatography, When pH of the protein of interest is lower than the pI then the protein will carry a net positive charge then Protein B and C will pass during unabsorbed fraction or during washing and in eluted fraction Protein A will come out.

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