Question 1 of 6: Polyacrylamide Gel Electrophoresis (PAGE)

Question 1: Polyacrylamide Gel Electrophoresis (PAGE) (20 marks)

Question text not reproduced: the examination questions are © Engineers and Geoscientists BC. Open the official past paper (linked at the top of this page) to read the question, then follow the worked solution below.

(a)(i) Why SDS Is Added

Sodium dodecyl sulfate is an anionic detergent that binds along the length of a denatured polypeptide backbone at a roughly constant mass ratio (~1.4 g SDS per g protein), coating every protein with a large, uniform negative charge that swamps out the protein's own native charge. It also unfolds the polypeptide into an extended, rod-like conformation, so shape differences between proteins are eliminated as well. With charge and shape standardized, electrophoretic mobility through the polyacrylamide sieve is governed almost entirely by chain length (molecular weight), which is exactly what makes SDS-PAGE useful as a size-separation and size-estimation technique — without SDS, migration reflects an uncontrolled mix of size, shape, and intrinsic charge (native PAGE).

(a)(ii) Why β-Mercaptoethanol Is Added

β-Mercaptoethanol (and DTT) is a reducing agent that cleaves covalent disulfide (S–S) bonds, both within a single polypeptide (intrachain, stabilizing tertiary structure) and between separate polypeptide chains (interchain, holding multi-subunit proteins together). Combined with heating, this fully denatures the protein and, for a multi-chain protein such as an antibody, dissociates it into its individual constituent chains. Reduction is added specifically to reveal a protein's true subunit composition and to ensure every chain runs at its own individual molecular weight rather than as part of a larger disulfide-linked complex.

(b) Why a Native Gel Was Chosen

eGFP and DsRed are visualized directly on a blue-light transilluminator, which relies on their intrinsic fluorescence — and fluorescence in this whole protein family depends on an internal chromophore that only forms and only fluoresces when the protein is folded into its correct, intact β-barrel tertiary (and, for DsRed, quaternary) structure. SDS and heat would denature both proteins, unfolding the barrel, exposing the chromophore to solvent, and abolishing fluorescence entirely — the technician would see two clean size-separated bands under white light stain, but nothing under the blue-light transilluminator. Running a native gel keeps both proteins folded and fluorescent throughout the run, so the bands can be located and compared directly by their own fluorescence with no additional staining step, which is the entire point of using fluorescent-protein reporters in the first place.

(c) Multiple Fluorescent Bands per Lane

Native PAGE does not force every copy of a protein into one uniform denatured state, so several distinct native species of the same protein can coexist in a sample and resolve as separate bands. Because native mobility depends on the actual size, shape, and net charge of whichever species is present — not on chain length alone — each one migrates to its own position within the lane.

In the gel actually printed as Figure 1, the multiple bands are in lane 1: eGFP gives a bright, far-migrating band (its ~33 kDa monomer) with a fainter band immediately above it, consistent with the weak self-association (dimer, ~65 kDa) that GFP variants are well known to show at the concentrations used for a gel. Lane 2 (DsRed) shows a single band on this gel — its obligate tetramer, assembled and matured uniformly enough that no dimer or free-monomer species is resolved.

The general reasons a lane may carry more than one fluorescent band are therefore: an equilibrium or incompletely assembled distribution across oligomeric states (monomer/dimer/tetramer), which for an obligately oligomeric protein such as DsRed can give tetramer plus lesser dimer and unassembled monomer if some copies have not fully matured or a fraction dissociates during handling; partially matured chromophore states, which fluoresce but differ slightly in charge; and minor conformational or charge (e.g. deamidation) variants of the same oligomer. Any of these can add further faint sub-bands.

[Figure not reproduced: Fig. 1 — Band pattern as printed in the source Figure 1: lane 1 (eGFP) carries two bands — a bright, far-migrating ~33 kDa monomer and a fainter ~65 kDa self-association dimer just above it — while lane 2 (DsRed) carries a single band that sits clearly above both lane-1 bands, the . See the official exam paper.]

(d) Are the Bands Where Expected?

Not simply by the stated per-chain molecular weights — and that is the key point of this question. eGFP (32.7 kDa) is essentially monomeric under native conditions, so its dominant fluorescent band is expected close to its stated ~33 kDa. DsRed, however, is stated as MW = 27.6 kDa per monomer, but DsRed does not exist as a free monomer under native, folded, fluorescent conditions: it assembles obligately into a homotetramer, so its dominant native species has an apparent mass around 4 × 27.6 ≈ 110 kDa — four times its stated per-chain weight, and some three and a half times eGFP's native monomer mass. This is exactly what the printed gel shows: lane 2's single DsRed band sits well above both of lane 1's bands, i.e. it has migrated the least far of anything on the gel. A technician who naively expected the smaller-monomer protein (DsRed, 27.6 kDa) to migrate farther than the larger-monomer protein (eGFP, 32.7 kDa) would be surprised: DsRed's dominant band should in fact run less far (higher apparent MW) than eGFP's, because native PAGE mobility tracks the assembled oligomer that is actually present, not the monomer molecular weight quoted for the polypeptide chain. Native mobility is also influenced by each protein's intrinsic net charge and overall shape (not just mass), so even the direction and spacing of migration can deviate further from a naive size-only prediction — this is precisely why native gels are read with the true oligomeric state and charge in mind, unlike SDS-PAGE where mobility tracks chain length alone.