Consider the table of four proteins below: Protein Size pI Size exclusion Charge
ID: 205519 • Letter: C
Question
Consider the table of four proteins below:
Protein
Size
pI
Size exclusion
Charge at pH 7.5
DEAE-elution
1
112 kDa
7.0
2
85 kDa
4.2
3
48 kDa
8.7
4
89 kDa
6.8
In what order would the proteins elute from a size-exclusion column? (from 1 = fastest to 4 = slowest).
Based on the pI of each protein, fill out the column in the table for charge at pH 7.5. Use these symbols: ++, +, -, --. Give a protein a double charge if its pI is more than 1 pH unit from the pH of the solution.
What proteins have the same charge as the column at pH 7.5 and so will pass through the column very quickly (all together, in the “void” volume)? Label those as “1” in the elution.
In what order would the remaining proteins elute from a DEAE anion exchange column when you wash with high concentration NaCl? Hint: lower charged proteins will tend to come off more easily à faster off.
Now, lead yourself in steps (b) – (d) again, using a CM cation exchange column where the initial binding is at pH 5.5 followed by a wash with high concentration NaCl.
To completely purify all proteins from each other, you plan to run a DEAE column followed by a CM column. Your boss asks you to reconsider. What is wrong with your plan? What should you do instead?
Protein
Size
pI
Size exclusion
Charge at pH 7.5
DEAE-elution
1
112 kDa
7.0
2
85 kDa
4.2
3
48 kDa
8.7
4
89 kDa
6.8
Explanation / Answer
In size exclusion chromatography, larger molecules move faster and elute first; smaller molecules enters the pores present in the beads, therefore they move slowly and elute lasts. So the order of elution is - protein 1 followed by protein 4 followed by protein 2 followed by protein 3
The isoelectric point (pI) is defined as the pH at which a protein has no net charge. When the pH > pI, a protein has a net negative charge and when the pH < pI, a protein has a net positive charge. Therefore, charge on proteins at pH7.5 -
Column with positive charge will tend to elute the more positively charged protein then less positively charged protein. Therefore, the order of elution will be Protein 3 followed by Protein 4.
DEAE has positive charge so it will bind tightly to the negative charge protein, after binding they can be eluted using either a pH or a salt gradient in the order Protein 1 followed by Protein 2
Using a CM cation exchange column, the order of protein elution will be protein 2 followed by protein 1 followed by protein 4 followed by protein 3