Suppose you have a mixture of five proteins listed in the table below: a) protei

Question

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

a) protein (a)       pI =4.6         mol.weight,kDa = 45

b) protein (b) pI =7              mol.weight,kDa =26.7

c) protein (c)       pI =4.9           mol.weight,kDa = 68.5

d)protein (d)        pI =6.4          mol.weight,kDa = 8.5

e)protein (e)        pI = 10.6         mol.weight,kDa =13

Indicate the order in which there proteins elute from a Bio-Gel p-10 gel-filtration column (starting with the on that elutes first). Is this the best resin to separate all the components of this mixture? Why? If not, what other Bio-Gel resin can you use to separate all the components of the mixture? This requires you look up some information about the size exclusion limits for each type of resin

2)You load this mixture on a cation exchange column. The buffer you use for this column is acetate buffer, pH 4.76.

2a) List the protein or proteins that will appear in the flow through (will not bind to the column)

2b) 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 1M. Indicate the order in which the proteins bound to the column will elute as the salt concentration increases.

c) Repeat the same procedure as in b. But now you use anion exchange column, and the buffer is TRIS (pH8). Answer the same questions as seen in i. and i i.

Explanation / Answer

Bio-Gel p-10 gel-filtration column has typical fraction range of 1,500-20,000 (Daltons). For globular molecules, fractionation ranges above 40,000 dalton. If the above mentioned proteins are globular particularly the proteins c, a & b then following would be the elution profile of the proteins. 'c' (68.5 kDa) elutes first then 'a' (45 kDa) followed by 'b' (26.7 kDa), 'e' (13 kDa) and finally 'd' (8.5 kDa).

The net charge on the molecule is affected by pH of its surrounding environment and can become more positively or negatively charged due to the gain or loss, respectively, of protons (H+). At a pH below the protein's pI, protein carries a net positive charge; above its pI it carries a net negative charge.

If the protein mix mentioned above is loaded on to the cation excange column in the presence of acetate buffer, pH 4.76, all the proteins except for protein 'a' gets net positive charge and bind to the cation excange column, and protein 'a' is found in the flow through along with some fraction of protein 'c'. The cation exchnage column bound proteins can be eluted by increasing the salt concentration and following would be the elution profile of the proteins. 'c' followed by 'd', 'b', and finally 'e' .

If the protein mix mentioned above is loaded on to the anion excange column in the presence of Tris buffer, pH 8.0, all the proteins except for protein 'e' gets net negative charge and bind to the cation excange column, and protein 'a' is found in the flow through. The anion exchnage column bound proteins can be eluted by increasing the salt concentration and following would be the elution profile of the proteins. 'b' followed by 'd', 'c', and finally 'a' .

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