Q.34
A mixture of four proteins of pIs 11, 7, 5 and 3 are loaded on a DEAE anion-exchange
column equilibrated with low ionic strength buffer of pH 8.
Which of the four proteins would be expected to be retained on the column?
Options:
(A) Protein with pI 11 but not the others
(B) Proteins with pIs 11 and 7 but not 5 and 3
(C) Proteins with pIs 7, 5 and 3
(D) Protein with pI 7 but not the others
DEAE (diethylaminoethyl) cellulose is a positively charged weak anion exchanger used to separate proteins based on net negative charge. At starting buffer pH 8 (low ionic strength), proteins with pI < buffer pH carry net negative charge and bind the column; proteins with pI > pH 8 (net positive) flow through unbound.
The correct answer is (C) Proteins with pIs 7, 5 and 3, as all three have pI < 8, making them negatively charged and retained on the DEAE resin.
DEAE Binding Rule: pI < Buffer pH = Negative Charge = Binds
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Column pH 8: DEAE positively charged (protonated amine groups)
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Proteins pI < 8: Net negative charge → electrostatic attraction → bind
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Proteins pI > 8: Net positive charge → repulsion → elute in flow-through
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Elution: Increasing salt gradient (NaCl) displaces bound proteins by charge competition
Protein Charge Analysis at pH 8
| Protein | pI Value | pH 8 – pI | Net Charge at pH 8 | Binds DEAE? |
|---|---|---|---|---|
| A | 11 | -3 | +3 (positive) | No |
| B | 7 | +1 | -1 (negative) | Yes |
| C | 5 | +3 | -3 (negative) | Yes |
| D | 3 | +5 | -5 (negative) | Yes |
Explanation of All Options
-
(A) Protein with pI 11 but not others
Wrong. pI 11 > pH 8 = positive charge = flows through. Others (pI 7,5,3) bind. -
(B) Proteins pI 11 and 7 but not 5 and 3
Incorrect. pI 11 doesn’t bind; pI 5,3 definitely bind (more negative). -
(C) Proteins pI 7, 5 and 3
Correct. All pI < 8 = negative charge = retained on DEAE column. -
(D) Protein pI 7 but not others
No. pI 5 and 3 even more negatively charged, bind more strongly.
Elution order (increasing NaCl): pI 7 (weakest binding) → pI 5 → pI 3 (strongest binding).
Biotech relevance: DEAE crucial for monoclonal antibody purification, plasmid DNA prep. Memory trick: “DEAE = Draws Everything Anionic (pI < pH).” pI 11 stays in supernatant; pI 7,5,3 stick to resin.
