20-Bio-B10 Biomechanical Device Design & Human Factors · December 2015
Nivaar worked solution (AI-drafted; not reviewed by a licensed engineer)
Paper format: National Exams, December 2015 — 04-Bio-B10 Analytical Biochemistry. Three hours, closed book, any non-communicating calculator. Six questions of equal value (20 marks each); five constitute a complete paper and only the first five appearing in the answer book are marked. All six are solved here, because this set is a study resource rather than an examination script. Every question is essay/descriptive (technique principle, interpretation of an instrument trace or image), with no numerical calculation on this sitting.
Reference texts (the books a candidate should have reviewed for this subject):
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.
Affinity chromatography purifies a target molecule by exploiting a highly specific, reversible, non-covalent binding interaction between the target and a complementary ligand that has been immobilized on an insoluble solid support (resin/matrix) packed into a column. When a crude mixture is passed over the column, only molecules capable of binding the immobilized ligand — ideally just the target — are retained, while everything else (the vast majority of the mixture's components, which have no meaningful affinity for that specific ligand) flows straight through unbound. The bound target is then selectively released by disrupting the binding interaction (e.g., a pH shift, a competing free ligand, or a change in ionic strength), yielding it in high purity, often in a single step and with a large fold-purification, because the separation is based on biological/chemical specificity rather than a bulk physical property like size or charge (which many co-eluting contaminants might share).
A full IgG antibody is a Y-shaped, ~150 kDa glycoprotein built from two identical heavy chains (~50 kDa each) and two identical light chains (~25 kDa each), held together by a combination of inter-chain disulfide bonds and non-covalent interactions. Each arm of the Y (a Fab, "fragment antigen-binding," region) pairs one light chain with the N-terminal portion of one heavy chain and is built from paired variable domains (VL and VH, whose hypervariable loops form the antigen-binding site) followed by constant domains (CL and CH1). The two heavy chains continue past a flexible, disulfide-bonded hinge region into the stem of the Y, the Fc ("fragment crystallizable") region, built from paired CH2/CH3 constant domains; the Fc stem is what is recognized by Fc receptors on immune cells and by complement, and is also the region typically bound by Protein A/Protein G affinity resins used to purify antibodies. N-termini sit at the tips of the two Fab arms (where antigen binds); the C-terminus sits at the base of the Fc stem.
Lectins are proteins that bind specific carbohydrate (sugar) structures with high specificity and are not themselves enzymes acting on the sugar. Immobilized on a solid support, they are used to capture glycosylated molecules — principally glycoproteins and glycolipids — by recognizing a particular sugar moiety on the attached glycan (for example, concanavalin A binds α-D-mannosyl/α-D-glucosyl residues, while wheat germ agglutinin binds N-acetylglucosamine/sialic acid residues). Lectin affinity chromatography is therefore used to enrich or purify specific glycoproteins (or glycoforms of a protein) from a complex mixture, based on their carbohydrate decoration rather than on protein sequence or antigenic identity.
Affinity purification follows a standard sequence of column operations. First, the column is equilibrated with a binding buffer that favours the target–ligand interaction. The crude sample is then loaded: as it passes through, the target binds the immobilized ligand while the great majority of unrelated proteins and other components have no affinity for it and pass straight through in the flow-through. The column is then washed with further binding buffer to rinse away unbound material and any weakly/non-specifically adsorbed contaminants, without disturbing the genuine, high-affinity target–ligand interaction. Finally, the target is eluted by deliberately disrupting that binding interaction — commonly by shifting pH, raising ionic strength, adding a chelating agent, or adding an excess of a free competing ligand — which releases the purified target from the matrix into the collected eluate, ready for neutralization/buffer exchange and any further polishing steps. The column can then be regenerated and re-equilibrated for reuse.
