You have a solution containing the following proteins Protein pI Mol. Weight, kD
ID: 36078 • Letter: Y
Question
You have a solution containing the following proteins
Protein
pI
Mol. Weight, kDa
Protein 1
4.2
125
Protein 2
5.7
32
Protein 3
9.6
10
Protein 4
3.2
45
Protein 5
12.0
60
1) You load this solution onto a DEAE anion exchange column at pH 5.7. Which proteins bind to your column? Briefly explain your answer.
2) You now load these same proteins onto a carboxymethyl cation exchange column at pH 7. Please list the proteins that do not bind to your column and appear in the effluent.
3) You load this same mixture onto a size exclusion column. Please indicate the order of elution.
Protein
pI
Mol. Weight, kDa
Protein 1
4.2
125
Protein 2
5.7
32
Protein 3
9.6
10
Protein 4
3.2
45
Protein 5
12.0
60
Explanation / Answer
1) DEAE- stands for diethylaminoethyl-, a tertiary amino group which will carry a positive charge except at very alkaline pH values. So your medium will be positively charged and therefore will bind proteins which carry a net negative charge.
If the protein is suspended in a buffer whose pH value is less than the protein's pI, the protein will pick up protons from the buffer and hence have a net positive charge. Conversely, if the protein is suspended in a buffer more alkaline than the protein's pI, the protein will lose protons to the buffer and become net negatively charged. So, protein 1 and 4 will bind.
2) carboxymethyl cation exchange column carries a net negative charge, and binds to those proteins which have a positive charge. Those proteins will have a positive charge whose pI value is less than pH of the medium(in this case 7). So, protein 5 will not bind and appear as effluent.
3) In a size exclusion column, the largest protein elutes first, So, order of elution will be :- 1,5,4,2,3
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