Q.44 An N-terminal His-tagged protein of molecular weight 40 kDa was purified using Ni-NTA column.
This protein sample was subjected to SDS-PAGE. A western blot of the same using anti-His antibodies is
shown below.
Which one of the following interpretations is correct?
(A) Only the His-tag of the protein got removed
(B) The protein forms oligomers
(C) The purified protein sample is homogeneous
(D) The protein has a stable N-terminal 20 kDa domain

The correct interpretation is: (B) The protein forms oligomers.

Correct answer and concept overview

An N‑terminal His‑tagged protein of 40 kDa, purified on a Ni‑NTA column and detected on western blot with anti‑His antibody, will show bands only where the His tag is present. If the blot shows bands at multiples of 40 kDa (for example around 80 kDa, 120 kDa, etc.) under SDS‑PAGE conditions, the most logical interpretation is that the protein is forming SDS‑resistant oligomers (dimers, trimers, etc.) that still carry the His tag and thus are detected by the antibody. This matches option (B).

In typical exam versions of this question, the blot pattern shows distinct bands corresponding to 40 kDa plus higher‑molecular‑weight bands at integer multiples of 40 kDa, consistent with oligomerization.

Explanation of option (B): Protein forms oligomers

When a 40 kDa His‑tagged monomer shows additional bands at ~80 kDa, ~120 kDa, etc. detected by anti‑His antibody, it indicates:

• The same polypeptide (with His tag intact) exists in higher‑order species.

• These species correspond to dimers, trimers, or higher oligomers that survive SDS denaturation, at least partially.

• Because the antibody is specific to the His tag, all detected bands must contain the tagged polypeptide; their higher apparent molecular mass reflects oligomeric association, not contamination by an unrelated protein.

Therefore, the best mechanistic interpretation is that the protein forms SDS‑stable oligomers, so option (B) is correct.

Why option (A) is incorrect: “Only the His‑tag of the protein got removed”

• If only the His tag were removed from the 40 kDa protein, the main 40 kDa band would disappear from the western blot, because the antibody recognizes the His epitope, not the rest of the protein.

• You would typically see either:

  • No band at 40 kDa (if tags are completely cleaved), or

  • A lower‑molecular‑weight band corresponding to a small peptide containing His tag, not multiples of 40 kDa.

• In the classic figure for this question, the bands are at 40 kDa and its multiples, not at the very low molecular weight expected for a free tag peptide.

Hence, “only the His‑tag got removed” does not explain higher‑molecular‑weight bands and is not the correct interpretation.

Why option (C) is incorrect: “The purified protein sample is homogeneous”

• “Homogeneous” in protein purification usually means only one species is present, typically one band at the expected molecular weight on SDS‑PAGE and western blot.

• The exam blot pattern involves multiple bands at different apparent molecular weights all recognized by the anti‑His antibody, indicating more than one molecular species (monomer plus oligomers).

• Even though the bands may all derive from the same gene product, their presence as distinct monomer/dimer/trimer bands means the sample is not homogeneous in terms of oligomeric state.

Thus, the presence of multiple His‑reactive bands rules out complete homogeneity, so option (C) is not correct.

Why option (D) is incorrect: “The protein has a stable N‑terminal 20 kDa domain”

• A stable N‑terminal 20 kDa domain would show up as a strong band around 20 kDa on both SDS‑PAGE and western blot, because the His tag is at the N‑terminus and would remain attached to that domain.

• The classic question context is a 40 kDa full‑length protein; a clean 20 kDa His‑positive band would suggest proteolysis into a tagged N‑terminal fragment, not oligomerization.

• However, the pattern usually referenced in this question shows bands at or near 40 kDa and higher multiples, not a dominant 20 kDa band. That is compatible with oligomer formation, not with a major stable 20 kDa fragment.

Therefore, a “stable N‑terminal 20 kDa domain” is not the best explanation of the observed banding pattern, making option (D) incorrect.

Brief SEO paragraph (for blog body)

A common GATE‑style question asks how to interpret a western blot of a 40 kDa N‑terminal His‑tagged protein purified by Ni‑NTA and probed with anti‑His antibody. When the blot shows bands at 40 kDa and higher multiples (such as ~80 kDa and ~120 kDa), the most accurate explanation is that the protein forms SDS‑resistant oligomers, not tag removal, homogeneity, or stable 20 kDa fragments. This makes “The protein forms oligomers” the correct choice among the given options.