Q.25 Proteins can be separated electrophoretically on the basis of net acidic and basic charge tlurough
1. Isoelectric focusing
2. Differential centrifugation
3. Countercurrent Immunoelectrophoresis
4. Pulse field gel electrophoresis
Correct Answer: 1. Isoelectric focusing
Proteins separate electrophoretically by net charge through isoelectric focusing (IEF), where they migrate in a pH gradient until reaching their pI (zero net charge).
Option Analysis
1. Isoelectric focusing
IEF establishes pH gradient via ampholytes; proteins move until net charge = 0 at pI. Acidic proteins stop at higher pH, basic at lower pH. Highest resolution for charge-based separation. Correct answer.
2. Differential centrifugation
Size/density-based sedimentation, not electrophoresis. Pellets nuclei (1000g), mitochondria (10,000g). No charge separation. [conversation context]
3. Countercurrent Immunoelectrophoresis
Immunological technique detecting antigen-antibody precipitation lines. Uses charge migration but separates immune complexes, not individual protein charges.
4. Pulse field gel electrophoresis
Size-based DNA separation via alternating fields preventing reptation limits. Horizontal agarose gels; irrelevant for proteins/net charge.
Proteins separated electrophoretically by net acidic basic charge through isoelectric focusing provides unmatched resolution in proteomics and 2D electrophoresis. This proteins separated electrophoretically net acidic basic charge isoelectric focusing guide targets GATE Life Sciences, CSIR NET biotech aspirants mastering separation techniques.
IEF Mechanism
Ampholyte-generated pH 3-10 gradient + electric field → proteins migrate to pI:
-
Acidic proteins (pI <7): Focus low pH regions (anode)
-
Basic proteins (pI >7): Focus high pH regions (cathode)
Resolution: ΔpI = 0.02 detectable. First dimension of 2DE.
Technique Comparison Table
| Method | Separation Basis | Charge-Based? | Protein Resolution |
|---|---|---|---|
| Isoelectric focusing | pI/net charge | Yes | Highest |
| Differential centrifugation | Size/density | No | Low |
| Countercurrent IEP | Immune complex | Indirect | Antigen-specific |
| Pulse field gel | DNA size | No | DNA only |
Proteins separated electrophoretically net acidic basic charge isoelectric focusing dominates charge fractionation.
Applications & Exam Tips
2D-PAGE first dimension; glycoprotein microheterogeneity; PTM analysis. Mnemonic: “IEF = pI Equalizer for acidic/basic proteins.” IPG strips > carrier ampholytes for reproducibility.
