3.
Protein X is composed of 700 amino acids. When resolved on denaturing PAGE it shows
a band corresponding to 130 kDa. The probable reason for such discrepancy is
a. Dimerization of the protein
b. Glycosylation of the protein
c. Phosphorylation of the protein
d. Acetylation of the protein

Correct answer: b. Glycosylation of the protein.

A 700-amino-acid protein is expected to have a molecular weight of roughly $700\times 110\text{ Da}\approx 77\text{ kDa}$, using the rule of thumb that an average amino acid residue is about 110 Da. Seeing a 130 kDa band on denaturing SDS-PAGE therefore suggests a large apparent increase in mass that is best explained by extensive glycosylation rather than simple small chemical modifications.​​

Introduction

When a 700 amino acid protein runs as a 130 kDa band on denaturing SDS-PAGE, the result looks puzzling—its apparent molecular weight is far higher than the ~77 kDa predicted from amino acid composition. Understanding why this discrepancy occurs is essential for CSIR NET and other life science exams, because it tests core concepts of protein mass estimation, SDS-PAGE principles, and post-translational modifications such as glycosylation.​​

Expected molecular weight of Protein X

• The average amino acid residue in a protein has a mass of about 110 Da (0.11 kDa).​

• For 700 residues: $700\times 0.11\text{ kDa}\approx 77\text{ kDa}$.​

So, the polypeptide backbone of Protein X is expected to be roughly 75–80 kDa, but SDS-PAGE shows a 130 kDa band, a difference of about 50 kDa that must be explained.​

Analysis of options

Option a: Dimerization of the protein

SDS-PAGE is a denaturing technique in which SDS binds along the polypeptide chain, unfolding the protein and imparting a uniform negative charge roughly proportional to length. Under standard “denaturing PAGE” (SDS-PAGE) conditions, non‑covalent dimers dissociate into monomers, so a simple dimerization through non‑covalent interactions would not persist and would not explain a single 130 kDa band for a 700-aa monomeric chain.​​

Furthermore, even if the protein formed a covalent dimer (e.g., via disulfide bonds), the apparent mass would be about $2\times 77\text{ kDa}\approx 154\text{ kDa}$, not 130 kDa, so the number does not match a straightforward dimer. Thus, option a is not the best explanation.​

Option b: Glycosylation of the protein (Correct)

Glycosylation is the covalent attachment of carbohydrate chains (glycans) to specific amino acids in proteins, typically Asn (N‑linked) or Ser/Thr (O‑linked), and glycans add substantial mass—often tens of kilodaltons in heavily glycosylated proteins. Importantly, glycans bind little or no SDS compared with polypeptide backbone, altering the charge-to-mass ratio and causing glycoproteins to migrate more slowly and thus appear at a higher apparent molecular weight on SDS-PAGE than predicted from sequence alone.​​

Because the observed mass (130 kDa) is much higher than the ~77 kDa backbone, a large added mass plus altered SDS binding and mobility is exactly what is expected for a strongly glycosylated protein, making glycosylation the most probable reason for the discrepancy. Therefore, option b is correct.​

Option c: Phosphorylation of the protein

Phosphorylation adds a phosphate group (~80 Da) per modified residue, which is very small compared with kilodalton-level changes; even multiple phosphorylation sites would only increase the mass by a few hundred daltons to at most a few kilodaltons. On SDS-PAGE, phosphorylation can cause subtle mobility shifts, sometimes visible as slight band shifts or a smear if hyperphosphorylated, but not a jump from ~77 kDa to 130 kDa.​

Thus, phosphorylation cannot account for such a large apparent increase in molecular weight, so option c is incorrect.

Option d: Acetylation of the protein

Acetylation typically adds an acetyl group (~42 Da) to lysine side chains or the N-terminus, again a very small mass change relative to the size of the protein. Even if many lysines were acetylated, the total added mass would remain in the low kilodalton range, far below the ~50 kDa discrepancy seen here.​

Acetylation does not typically shift SDS-PAGE bands by dozens of kilodaltons, so it is not a plausible explanation for a 700 amino acid protein appearing at 130 kDa; therefore, option d is also incorrect.

Why glycosylation changes apparent SDS-PAGE mass

Glycoproteins often migrate anomalously on SDS-PAGE because:

• Carbohydrate chains increase the hydrodynamic size and do not bind SDS efficiently, so the SDS:mass ratio is lower than for unglycosylated proteins.​​

• As a result, glycosylated proteins move more slowly through the gel matrix and appear as higher apparent molecular weight bands, sometimes smeared if glycosylation is heterogeneous.​​

In exam questions, a large gap between “calculated from sequence” and “observed on SDS-PAGE” for a secreted or membrane protein is a classic clue pointing towards glycosylation as the cause.