Q.2 Two-dimensional electrophoresis resolve the proteins on the basis of :
- (1) Net mass and osmotic pressure
- (2) Net viscosity and volume
- (3) Net charge and mass
- (4) Net charge and volume
The correct answer is option (3): Net charge and mass.
Two-dimensional electrophoresis (2DE) separates proteins first by isoelectric point (net charge) via IEF, then by molecular weight (mass) via SDS-PAGE.
Option Explanations
(1) Net mass and osmotic pressure – Incorrect
Osmotic pressure irrelevant to electrophoresis; separation uses electric fields, not osmosis.
(2) Net viscosity and volume – Incorrect
Viscosity/volume don’t drive electrophoretic migration; proteins separated by charge/mass in electric field.
(3) Net charge and mass – Correct
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1st dimension (IEF): Proteins migrate to pI (net charge = 0) in pH gradient
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2nd dimension (SDS-PAGE): SDS coats proteins (charge ∝ length), separated by mass through gel matrix
(4) Net charge and volume – Incorrect
SDS-PAGE separates by mass, not volume. Stokes radius correlates with mass under denaturing conditions.
2DE Mechanism
text1st Dimension: IEF (horizontal)
Protein → pI position where net charge = 02nd Dimension: SDS-PAGE (vertical)
SDS-Protein complex → migrates ∝ 1/mass through polyacrylamide pores
Result: Protein spots at (pI, MW) coordinates; >3000 proteins resolvable
Why Charge + Mass?
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Orthogonal separation: Independent parameters maximize resolution
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IEF: Charge-based (pI 3-10 range)
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SDS-PAGE: Mass-based (5-200 kDa range)
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Spot pattern: Unique (pI,MW) signature per protein
2D Electrophoresis Protein Separation Principle
Two dimensional electrophoresis net charge mass separation is cornerstone proteomics technique for GATE Life Sciences. First dimension (IEF) resolves by isoelectric point, second (SDS-PAGE) by molecular weight creating high-resolution protein maps.
First Dimension: Isoelectric Focusing (Net Charge)
Proteins migrate in pH gradient until reaching pI (net charge = 0):
textLow pI (-) ←───────── pH Gradient ──────────→ High pI (+)
Acidic proteins Basic proteins
Second Dimension: SDS-PAGE (Mass)
SDS denatures + uniform negative charge ∝ polypeptide length:
textSmall MW ──────────────→ Large MW ↓
Fast migration Slow migration
Resolution Power Comparison
Technique Resolution Basis 2DE 3000+ spots Charge + Mass 1D SDS-PAGE 50-100 bands Mass only IEF alone 100-200 bands Charge only Applications in Research
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Proteomics: Differential expression (disease vs normal)
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Post-translational modifications: Charge shifts
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Protein isoforms: Mass/charge variants
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Biomarker discovery: Spot intensity changes
Exam Memory: “IEF = Charge, SDS = Size” → Net charge + mass
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