You have a mixture of 5 Proteins. Their relation characteristic pI value are not

Question

You have a mixture of 5 Proteins. Their relation characteristic pI value are noted in relation to the a working pH of 6.5 for the experiment: Suppose that your column (sec questions below for both cation and anion column) is equilibrated and being eluted at pH 6.5 (the working pH is 6.5). The mixture of proteins is loaded onto the column at a low ionic strength (low salt). Some of the proteins 1-5 will bind and some will not. Some will be in solid volume and some will bind to the column depending which type of column is used. By washing the column with a gradient of buffer of increasing ionic strength or salt concentration the proteins that have bound can be eluted after the void volume proteins have been eluted. Draw and label an elution profile (see slides from both gel filtration and ion exchange to sec what a profile looks like) with #1, #2, #3, #4, and/or #5, based on the expected order of elution of proteins #1-5 from 1) a cation exchange column, and from 2) an anion exchange column, at pH 6.5. Do all proteins bind both columns? Explain for both column types. What proteins do not bind each of the columns under these buffer conditions for each exchanger and hence are eluted in the void volume? Explain for both types of column.

Explanation / Answer

For the gven proteins

A. the elution order for the proteins on,

cation exchange resin would be : (First to elute) : 1 < 2 < 3 < 4 < 5 (last to elute)

Anion exchange resin would be : (first to elute) : 5 < 4 < 3 < 2 < 1 (last to elute)

B. The proteins bind to the column according to its net charge at pH 6.5. If the cation exchnage column is used, the more the positive the protein is, stringer would be the binding and delayed elution from the column would be seen for the protein. Similarly for the anion exhchange column, the more the negative the protein charge is, stronger would be the binding of protein would be on the column and delayed elution would be for the protein from the column.

C. The proteins that have PI = pH 6.5, would have a neutral charge and would not bind to eigther of the column at this pH.

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