Q.14 Isoelectric focusing allows seperation of the proteins according to their-
1.pH
2. pl
3. pKa
4. pKb
Isoelectric focusing separates proteins based on their isoelectric point (pI), the pH at which they carry zero net charge. The correct answer is option 2: pI.
Technique Overview
Isoelectric focusing (IEF) uses a pH gradient gel and electric field, where proteins migrate until reaching their pI and stop moving due to charge neutrality. This provides high-resolution separation superior to SDS-PAGE for charge-based differences.
Option Analysis
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1. pH: Incorrect. pH is the medium property creating the gradient; proteins don’t separate by bulk pH but by individual response to it.
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2. pI: Correct. At pI, protein net charge = 0 (from Henderson-Hasselbalch balance of ionizable groups); migration ceases here.
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3. pKa: Incorrect. pKa measures acid dissociation constants of specific residues; IEF uses overall pI (average of relevant pKas).
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4. pKb: Incorrect. pKb relates to base dissociation (rarely used for proteins); irrelevant to amphoteric protein separation.
Options 1,3,4 fail as they confuse environmental pH, residue-specific constants, or base metrics with the key parameter: protein’s unique pI.
Isoelectric focusing allows separation of proteins according to their isoelectric point (pI), not pH, pKa, or pKb. This GATE Life Sciences guide decodes IEF protein separation principles, compares options, and equips competitive exam aspirants with high-resolution electrophoresis knowledge.
IEF Mechanism
Proteins (zwitterions) gain + charge below pI, – charge above, migrating in electric field through ampholyte-generated pH gradient (pH 3-10 typical). At pI: ∑charges = 0, focusing into sharp bands (resolution <0.01 pH units).
| Parameter | Role in IEF | Separates By? | Correct? |
|---|---|---|---|
| pH | Gradient medium | No—environment | No |
| pI | Protein’s zero-charge pH | Yes—unique per protein | Yes |
| pKa | Residue dissociation | No—contributes to pI | No |
| pKb | Base strength | No—protein irrelevant | No |
Why pI Dominates
IEF’s “focusing” counters diffusion via opposing electrophoretic forces at pI. 2D-PAGE pairs IEF (1st dim., pI) + SDS-PAGE (2nd dim., MW) for proteome mapping. Variants: IPG strips (stable gradients).
Exam Mastery
Mnemonic: Isoelectric Focusing = Identifies p****I. Distinguish: SDS-PAGE (size), Native-PAGE (charge/shape), IEF (pI). Practice calculating pI: average pKa of ionizable groups (Asp/Glu low pI; Lys/Arg high).
