Q.No 32. A globular protein of molecular weight 50 kDa exists as a mixture of monomers and dimers in solution. The most appropriate technique for the separation of these two forms of the protein is
**Gel filtration chromatography separates the 50 kDa globular protein’s monomers (∼25 kDa) and dimers (∼50 kDa) based on size differences in solution. **
Option Analysis
Thin Layer Chromatography (A) separates compounds by adsorption on a thin adsorbent layer, mainly for small molecules like amino acids. It lacks resolution for large proteins like 50 kDa forms due to poor handling of macromolecules in native states.
Ion Exchange Chromatography (B) uses charge differences on resins to bind and elute proteins. Monomers and dimers of the same protein have identical charges, preventing effective separation.
Gel Filtration Chromatography (C) employs porous beads where dimers elute first (excluded from pores) and monomers later (enter pores), ideal for size-based separation of oligomers.
Paper Chromatography (D) partitions small molecules like amino acids via paper’s cellulose. It fails for proteins due to low resolution and denaturation issues.
Correct Answer
(C) Gel filtration chromatography best isolates monomers from dimers without denaturing the native protein mixture.
Gel filtration chromatography, also known as size exclusion chromatography, excels at separating protein monomers and dimers based on molecular size differences. For a 50 kDa globular protein existing as monomers (∼25 kDa) and dimers (∼50 kDa) in solution, this technique uses porous beads to fractionate forms efficiently.
Why Gel Filtration for Protein Separation?
Larger dimers travel faster through the column, excluded from bead pores, while smaller monomers enter pores for longer paths and later elution. This non-denaturing method preserves native structures, unlike electrophoresis.
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Ideal for oligomeric analysis in biochemistry research.
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Common in CSIR NET Life Sciences for protein purification questions.
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Buffers like 0.1 M NaCl minimize unwanted interactions.
Comparing Chromatography Techniques
| Technique | Basis | Suitability for 50 kDa Protein Monomers/Dimers |
|---|---|---|
| Thin Layer Chromatography | Adsorption | Poor; for small molecules, low protein resolution |
| Ion Exchange Chromatography | Charge | Ineffective; similar charges |
| Gel Filtration | Size | Excellent; direct separation |
| Paper Chromatography | Partition | Unsuitable; for amino acids |
Ion exchange fails as charge is identical, while planar methods like TLC/paper lack capacity for proteins.
CSIR NET Exam Insights
This question tests Unit 13 (Methods in Biology), emphasizing size-based techniques for native proteins. Practice similar problems on protein molecular weight determination.
