1. Listed below is a mixture of 5 proteins and their characteristics. In each ca
ID: 192357 • Letter: 1
Question
1. 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). Letter Protein code Isoelectric point (pl) Molecular Weight A Ovalbumin Myoglobin (kDa) 45 16.7 68.5 8.5 13 4.6 C Serum albumin E Cytochrome c a) Indicate the order in which these proteins will elute from gel-filtration column. Start with the 4.9 6.4 10.6 Ubiquitin one that elutes first using the letter code provided. b) You load this same mixture on a cation exchange. The buffer used for elution san 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. 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.OM. 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
Ans.a). The order of proteins eluting from gel filtration chromatography- C-A-B-E-D. By gel filtration chromatography, a mixture of proteins is separated by their size or molecular weight. It is performed in a column with very small porous beads with different pore sizes. During separation,larger molecules elute first as they cannot enter the pores, whlie the smaller molecules retain foe longer time.
b). cation exchange chromatography is a variant of ion exchange chromatography, which is used to separate molecules having net positive surface charges from a mixture by using a negatively charged ion exchange resin. If pH of a protein is less than its pI, it will move towards negative charge and vice versa. According to pI of these proteins, at pH 4.6, proteins B, D, and E will bind with the anionic ion exchange resin. Protein C will bind weakly, while protein A will not bind with the resin as its pI is equal to pH of the solution buffer and it will carry no charge.
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