20-Bio-B10 Biomechanical Device Design & Human Factors · May 2015
Nivaar worked solution (AI-drafted; not reviewed by a licensed engineer)
Paper format: National Exams, May 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) rather than numerical, except Question 2(d), which asks for a short exponential-growth calculation from PCR cycle theory.
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.
The standard, simplest choice is to stain the finished gel with Coomassie Brilliant Blue (R-250): the gel is fixed (methanol/acetic acid), soaked in Coomassie dye, then destained until the background clears, leaving the protein bands stained blue. Coomassie binds non-specifically to basic and aromatic side chains and is sensitive down to roughly 50–100 ng of protein per band — more than adequate for the microgram-scale loads typical of an affinity-capture check. If greater sensitivity were needed (e.g. to detect a trace contaminant), a silver stain (sensitive to 1–10 ng) could be used instead, at the cost of a longer, less quantitatively linear protocol. Because the two bands here are only being checked for identity and purity (not being sequenced or blotted), a general protein stain such as Coomassie is explained as sufficient; a Western blot with an anti-target or anti-antibody (anti-Fc) probe would be the appropriate follow-up only if the identity of a specific band needed to be confirmed rather than inferred from its apparent molecular weight.
SDS coats every polypeptide with a uniform negative charge roughly proportional to its length, so in SDS-PAGE all proteins run toward the positive electrode purely by size (smaller proteins thread through the polyacrylamide mesh faster and migrate farther). The loading wells therefore sit at the cathode (−) end of the gel, and the anode (+) is at the far end where the smallest species end up. Lane 1 below is a molecular-weight marker (ladder) run alongside the sample lane; the 150 kDa suspected-antibody band runs close to its position in the ladder (having migrated only a short distance, consistent with a large protein), while the 30 kDa target band has migrated much farther, consistent with the ladder's 37–25 kDa bracket.
The size of each band is determined from a calibration curve built from the marker lane: the migration distance of each marker band is measured from the well, and log₁₀(molecular weight) is plotted against migration distance — over the useful range of a given gel percentage this relationship is closely linear. The unknown bands' migration distances are then measured on the same gel and read off (interpolated on) that log-linear calibration line to give their apparent molecular weights: here, roughly 150 kDa and 30 kDa, consistent with an IgG-class antibody (~150 kDa intact) and the stated 30 kDa target.
DTT and β-mercaptoethanol are reducing agents that break intermolecular and intramolecular disulfide bonds; combined with heating (which denatures secondary/tertiary structure and helps SDS coat the polypeptide backbone uniformly), this converts a multi-subunit or disulfide-linked protein into its individual, fully denatured polypeptide chains. A full-length IgG antibody is held together by disulfide bonds into two identical heavy chains (~50 kDa each) and two identical light chains (~25 kDa each) (~150 kDa total, intact); reducing SDS-PAGE therefore resolves the single 150 kDa band into two new bands at roughly 50 kDa and 25 kDa. The 30 kDa target polypeptide, by contrast, was stated to contain no cysteine residues at all — it has no disulfide bonds to break, so reduction and heating cannot change its apparent size, and it should still run at 30 kDa, unchanged in intensity and position.
This is itself the confirmatory experiment: if the 150 kDa band really is the antibody, reduction must split it into the classic ~50/~25 kDa heavy/light-chain pair; if it were instead a non-disulfide-linked 150 kDa contaminant, reduction and heating would leave it as a single unchanged band. Seeing the heavy/light-chain pair appear — and the 30 kDa target remain exactly where it was — is the direct evidence that the second band was indeed the capture antibody and that the target protein has been successfully purified.
