Question

NOTES:

To separate on a size-exclusion column, differences in MW need to be greater than 10%.

To separate proteins by charge, the difference in pI must be at least 0.5 pH unit.

Consider the table of four proteins below:

Protein	Size	pI	Size exclusion	Charge at pH 7.4	DEAE-elution
1	49 kDa	6.8
2	51 kDa	8.1
3	53 kDa	6.4
4	99 kDa	8.3
5	106 kDa	3.8
6	143 kDa	7.9

(a) In what order would the proteins elute from a size-exclusion column? (from 1 = fastest to 6 = slowest). Some proteins can’t be separated from each other! Show this by including them in parentheses (1 > (2,3) > (4,5) > 6, for example).

(b) What is the charge on a DEAE column at pH 7.4?

(c) Based on the pI of each protein, fill out the column in the table for a charge at pH 7.4. Use these symbols: ++, +, -, --. Give a protein an extra charge for each FULL pH unit that its pI is different from the pH of the solution. Example. If the pH were 4.2, a protein with pI 6.3 would be + +.

(d) What proteins have the same charge as the column at pH 7.4 and so will pass through the column very quickly (all together, in the “void” volume)? Label those as “1” in the elution.

(e) 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  faster off. But see the NOTES above!

(f) Are all your proteins separated from each other now? If yes, you’re done. If no, explain why you haven’t done your job and separated them! (g) Now, lead yourself in steps (b) – (d) again, using a CM cation exchange column where the initial binding is at pH 7.0 followed by a wash with high concentration NaCl.

(h) 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?

Answer 1

I have already answered this before. I cannot have two of the same answers so I had to delete my answer here. So please excuse me and please find the answer here: https://www.HomeworkLib.com/homework-help/questions-and-answers/notes-separate-size-exclusion-column-differences-mw-need-greater-10-separate-proteins-char-q40376475

Thank you..