Suppose you have a mixture of five proteins listed in the table below. I. Indica

Question

Suppose you have a mixture of five proteins listed in the table below.

I. Indicate the order in which order these proteins will elute from a gel-filtration column (starting with the one that elutes first). You can use letters A-E (see table) for simplicity.

II. You load this mixture on a cation exchange column (i.e. column that bears negatively charged groups). The buffer you use for this column is acetate buffer, pH 4.76. List proteins that will appear in the flow-through (i.e. will not bind to the column).

III. In order to elute those proteins that are immobilized on the column, you then apply a linear salt gradient, with NaCl concentration gradually increasing from 0 to 1 M. Indicate the order in which the proteins bound to the column will elute as the salt concentration increases.

IV. You repeat the same procedure as in (b) but now you use anion exchange column (that bears positively charged groups), and the buffer is TRIS (pH 8.0). Answer the same questions as in (b).

Weight kDa pl Mol kDa Protein A Ovalbumin 4.6 45 B Myoglobin 7 16.7 C Serum albumin 4.9 68.5 D Ubiquitin 8.5 6.4 10.6 13 LE Cytochrome c

Explanation / Answer

1. C, A, B, E, D

2. A, C (In the latter case, the difference between pH 4.76 and pI 4.9 is too small, so serum albumin will also be in the flow-through).

3. D, B, E

4. Flow through: E Bound proteins elute as: B, D, C, A

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