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?
-
ASDS–PAGE electrophoresis alone
-
BGel filtration (size-exclusion) chromatography alone
-
CCombine SDS–PAGE (subunit MW) with gel filtration (native MW) to infer subunit number
-
DIsoelectric focusing alone
-
EA single native PAGE run without standards
Answer
Correct Answer: Combine SDS–PAGE (subunit MW) with gel filtration (native MW) to infer subunit number
Explanation
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:
1) Run SDS–PAGE → determine subunit masses (e.g., 25 kDa and 30 kDa).2) Run gel filtration → determine native complex (e.g., ~110 kDa).3) Compare: 110 kDa vs subunits implies likely stoichiometries (e.g., 2 * 25 + 2 * 30 = 110 → heterotetramer).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:
a) Reveals subunit MW but not intact native mass.b) Reveals native mass but not individual subunits.d) Resolves by pI, not mass; insufficient for stoichiometry alone.e) Lacks calibration and denatured subunit information.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.