Q.16 The molecular weight of a protein as determined by native PAGE is 400 kDa . This protein when run
on a non-reducing SDS-PAGE gave a band of 200 kDa , and on a reducing SDS-PAGE, gave a band of 100
kDa . The protein has
(A) four subunits of which two sets are linked by two disulfide bridges
(B) four subunits which are linked by four disulfide bridges
(C) two subunits only and none are linked by disulfide bridges
(D) two subunits which are linked by disulfide bridges
The correct answer is (A) four subunits of which two sets are linked by two disulfide bridges.
PAGE Techniques
Native PAGE preserves the protein’s quaternary structure, so the 400 kDa band indicates the full native molecular weight. Non-reducing SDS-PAGE denatures the protein with SDS but keeps disulfide bonds intact, yielding a 200 kDa band that represents dimers held by disulfides. Reducing SDS-PAGE adds agents like DTT or β-mercaptoethanol to break disulfide bonds, producing 100 kDa monomers.
Option Analysis
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(A) Correct: Four 100 kDa subunits form two disulfide-linked dimers (200 kDa each); non-covalent interactions hold the dimers into the 400 kDa native tetramer.
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(B) Incorrect: Four disulfide bridges linking all four subunits would show one 400 kDa band in non-reducing SDS-PAGE, not 200 kDa.
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(C) Incorrect: Two subunits without disulfides would show 200 kDa in both non-reducing and reducing SDS-PAGE, not 100 kDa in reducing.
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(D) Incorrect: Two subunits linked by disulfides would show 200 kDa native (or close), one 200 kDa in non-reducing, and 100 kDa in reducing, mismatching the 400 kDa native.
In protein molecular weight determination using native PAGE (400 kDa), non-reducing SDS-PAGE (200 kDa), and reducing SDS-PAGE (100 kDa), the results indicate a tetrameric protein with specific disulfide linkages—ideal for CSIR NET Life Sciences preparation.
Key Concepts
Native PAGE maintains quaternary structure for accurate native molecular weight (400 kDa here). SDS-PAGE denatures proteins for size-based separation; non-reducing keeps disulfides (200 kDa dimers), while reducing breaks them (100 kDa subunits).
Structure Insight
The protein comprises four identical 100 kDa subunits. Two pairs form disulfide-linked dimers (two bridges per pair), associating non-covalently into the 400 kDa tetramer.
Exam Relevance
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Matches option (A) precisely.
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Common CSIR NET trap: Confusing dimer vs. tetramer or disulfide count.
