Protein pMolecular Weight (kDa) 3.1 6.9 10.3 7.1 8.6 265 93 96 43 189 You load t
ID: 200150 • Letter: P
Question
Protein pMolecular Weight (kDa) 3.1 6.9 10.3 7.1 8.6 265 93 96 43 189 You load the protein mix onto a cation exchange column at pH 5. Next, you apply a "washing" step by passing through buffer at pH 5. Finally, for your elution step, you apply a pH gradient starting from pH 2.0 to pH 13.0. (A gradient buffer system allows you to gradually and continuously change the pH of vour mobile phase starting from pH 2 up to pH 13). Indicate the order in which proteins will exit / elute from the column during the elution step. Please explain your answer. You load this same mixture onto a size exclusion column. Please indicate the order of elution.Explanation / Answer
Ion exchange chromatography separates the molecules based on the charge present on the molecules. It uses the cation exchange resins such as CM-cellulose or anionic exchange resins such as DEAE cellulose to separate the molecules. Depending upon the type of charge on molecule they bind to the resins and they are eluted by the elution buffers.
The isoelectric point is the point at which protein exists as a neutral form. When the pH of solution less than the pI of protein then it exist as a cation and When the pH of solution greater than the pI the protein then it exists as an anion and when pI equals to pH then protein acid exist as neutral form.
S.No.
protein
pI
Molecular weight
1
A
3.1
265
2
B
6.9
93
3
C
10.3
96
4
D
7.1
43
5
E
8.6
189
1) When we apply pH gradient starting from the pH2.0 to pH 13. The protein is eluted from the column when the pH of gradient equal to pI of the solution. According to this information, the order of elution will be the
(First eluted) A>B>D>E>C (eluted last)
2) Size exclusion chromatography separates the proteins based on the size. When we place a sample of a mixture of proteins, depending upon the fractionation limit the small molecules will enter into the internal pores of the gel beads and they need to migrate along the path for their elution so eluted very slowly but bigger proteins cannot enter into the internal pores and eluted first. Based on this information order of elution is
(Eluted First) A>E>C>B>D (eluted last)
S.No.
protein
pI
Molecular weight
1
A
3.1
265
2
B
6.9
93
3
C
10.3
96
4
D
7.1
43
5
E
8.6
189
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