Listed below is a mixture of 5 proteins and their characteristics. In each case,

Question

Listed below is a mixture of 5 proteins and their characteristics. In each case, make sure to provide an explanation as to your choice(s). 1. Letter Protein code Isoelectric point (pl) Molecular Weight (kDa) 45 16.7 68.5 8.5 13 Ovalbumin 4.6 Myoglobin Serum albumin Ubiquitin Cvtochrome c 4.9 6.4 10.6 a) Indicate the order in which these proteins will elute from gel-filtration column. St tart with the one that elutes first using the letter code provided. You load this same mixture on a cation exchange. The buffer used for elution is an acetate buffer, pH 4.76. List the letter codes for proteins that don't bind to column and ultimately up in the flow-through. b) end linear salt gradient is applied to the cation exchange column above to elute the remaining bound proteins. The gradient used is NaCI with increasing concentration from 0.0 to 1.0 M. Indicate the order in which the proteins bound to the column will elute as the salt concentration increases. Remember to use the letter codes provided. c) A d) Next, you set up an anion exchange column and use the buffer Tris, pH 8.0. List protein(s) that will appear in the wash Your final mission is to study Cytochrome c separately from the other proteins. Based on the results of the experiment you preformed on the mixture, devise a single step procedure to separate it from the mixture of proteins. e)

Explanation / Answer

a) C, A, B, E, D
b)A, C ( serum albumin will also be in the flow-through as In the latter case, the difference between pH 4.76 and pI 4.9 is too small,).
c)D, B, E
d)Flow through: E
Bound proteins elute as: B, D, C, A
e)use anion exchange column : cytochrome c will be in the flow-through and a combination of size-exclusion chromatography and ion exchange can also be used.

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