I need some help with c, d and e Listed below is a mixture of 5 proteins and the
ID: 192582 • Letter: I
Question
I need some help with c, d and e 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 Ovalbumin Myoglobin (kDa) 45 16.7 4.6 C Serum albumin 4.9 6.4 10.6 Ubiquitin E Cytochrome c 8.5 13 a) Indicate the order in which these proteins will elute from gel-filtration column. Start 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 end up in the flow-through. b) A 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) 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)
In gel filtration chromatography, the larger proteins elute first since they do not enter the gel matrix.
The order of elution is CABED
(b)
At pH of 4.76, net charge on the proteins will be
A= slightly negative
B= positive
C = slightly positive
D= positive
E= highly positive
At 4.76, protein A will elute
(c)
As the salt concentration increase the following proteins will elute out in this order
CDBE
(d)
At pH 8.0
A= positive
B= slightly positive
C = positive
D= positive
E= negative
The protein that would appear in wash is E
(e)
Use Tris buffer of pH 8 and anion exchange column. Under these conditions only the cytochrome c is negatively charged and would elute out without binding to the anion exchange column.