Determining subunit stoichiometry — Which combination of methods lets you find both subunit molecular weight and the number of subunits in a protein's quaternary structure?

Introduction:Determining quaternary structure often requires knowing both the mass of the intact native assembly and the masses of its individual subunits. Integrating orthogonal separation data provides a practical route to subunit stoichiometry.

Given Data / Assumptions:

• SDS–PAGE estimates individual polypeptide molecular weights under denaturing conditions.
• Size-exclusion (gel filtration) estimates the native molecular weight of the intact complex.
• Subunit count ≈ native MW / dominant subunit MW (accounting for heterogeneity if present).

Concept / Approach:Use SDS–PAGE to identify subunit sizes and relative band intensities; use gel filtration to estimate the native complex mass. The ratio provides an integer consistent with known subunit composition, validated against standards and known oligomeric forms.

Step-by-Step Solution:

Verification / Alternative check:Cross-check with native PAGE or mass spectrometry. Non-reducing SDS–PAGE can reveal disulfide-linked oligomers complementing gel filtration data.

Why Other Options Are Wrong:

Common Pitfalls:Interpreting anomalous elution due to shape or glycosylation as true mass; ignoring hetero-oligomer composition when dividing masses.

Final Answer:Combine SDS–PAGE with gel filtration.