Biochemistry Techniques

Affinity Chromatography (Ni-NTA / His-tag)

Why it works (first principles)

This is arguably the single most important protein purification technique in IBO practicals - it appeared in 2016, 2018, and 2023. The principle is molecular recognition: six consecutive histidine residues (the “His-tag”) at one end of the protein coordinate Ni2+ ions that are immobilised on the chromatography resin (NTA = nitrilotriacetic acid). Because most native E. coli proteins lack stretches of histidine, only the His-tagged target protein binds tightly.

Contaminant proteins are washed away with a low-imidazole wash buffer. The target is then released (“eluted”) with a high-imidazole buffer - imidazole has an imidazole ring structurally similar to histidine’s side chain, so it competes for the nickel binding sites and displaces the protein.

Workflow (IBO style)

1. Equilibrate the column with lysis buffer - this removes storage ethanol and conditions the resin.
2. Load clarified bacterial lysate onto the column.
3. Wash extensively with wash buffer (low imidazole or standard lysis buffer) to remove contaminants.
4. Elute with elution buffer into successive fractions (usually 2-3 tubes).
5. Never let the resin run dry. This is the single most important rule. A dry resin collapses irreversibly and loses all binding capacity.

Exam traps

• Letting the resin dry between steps (catastrophic - irreversible).
• Incomplete washing - contaminated elution fractions.
• Collecting elution drops into the wrong tubes.
• Adding buffers too roughly, which disturbs the resin bed and reduces separation quality.

Bradford Protein Assay

Why it works (first principles)

Coomassie Brilliant Blue G-250 exists in three colour forms depending on its protonation state. The anionic (blue) form binds to basic and aromatic amino acid residues in proteins, and this binding stabilises the blue form, shifting the absorbance maximum to 595 nm. The more protein present, the more dye shifts to the blue form, and the higher the A595.

Workflow

1. Prepare a BSA standard series (typically 0-0.6 mg/mL in 7 tubes).
2. Dilute your unknown samples into the linear range of the assay.
3. Add Bradford reagent to each standard and unknown, incubate 5 minutes in the dark.
4. Read absorbance at 595 nm.
5. Plot A595 vs [BSA] for the standards, draw the best-fit line through the linear portion.
6. Interpolate the unknowns from the standard curve.
7. Multiply by the dilution factor to get the original concentration.

Typical BSA standard series (IBO 2018 and 2023):

The Bradford assay appeared in the 2018 and 2023 IBO exams, both times as part of the “Purify then Quantify then Assay” integrated workflow.

Exam traps

• Bubbles in microplate wells scatter light and inflate absorbance readings.
• Light exposure during the 5-minute incubation degrades the dye-protein complex.
• Using the non-linear high-concentration region of the standard curve for interpolation.
• The most common mistake: forgetting the dilution factor when back-calculating the original protein concentration.

Plot your own standard curve – enter BSA concentrations and absorbance values, then interpolate unknowns:

Need to calculate dilution volumes? Use this quick calculator:

Continuous Enzyme Assays

Why they work (first principles)

In a continuous assay, you follow the appearance or disappearance of a chromophore in real time. The most common chromophore in IBO practicals is NADH (absorbs at 340 nm). As the enzymatic reaction proceeds, NADH is either produced (in oxidation reactions, like ADH) or consumed (in coupled assays, like PK/LDH), and the absorbance changes proportionally.

The initial rate (the linear portion of the progress curve) gives you $v_0$, the velocity at the starting substrate concentration. By varying [S] and measuring $v_0$ at each concentration, you build a Michaelis-Menten curve and extract $K_M$ and $V_{\max}$.

General workflow

1. Prepare a master mix containing buffer, cofactors, and any coupling enzymes.
2. Add substrate (or enzyme) last to start the reaction.
3. Record absorbance at short intervals (every 15-30 seconds).
4. Plot A vs time, identify the linear region, and calculate the slope (= initial rate).
5. Convert the slope to velocity using Beer-Lambert:

6. Vary [S] and repeat to generate a Michaelis-Menten plot.
7. Extract $K_M$ (the [S] at half-$V_{\max}$) and $V_{\max}$ either graphically or via linear transformation (Hanes-Woolf: plot [S]/$v_0$ vs [S]; slope = 1/$V_{\max}$, x-intercept = $-K_M$).

ADH Assay (IBO 2022)

Alcohol dehydrogenase catalyses:

The increase in A340 directly reports NADH production and hence ADH activity.

Activity formula (from the 2022 exam):

where $V_1$ = total assay volume (3.0 mL), $V_2$ = volume of enzyme added (0.1 mL), and $\Delta t$ is in minutes.

The 2022 exam also tested active-site modifier effects: students pre-incubated ADH with EDTA (a metal chelator that strips out the essential Zn2+ cofactor) or PCMB (p-chloromercuribenzoate, which covalently modifies cysteine -SH groups). The results allowed students to deduce which residues and cofactors are essential for catalysis:

This tells you that yeast ADH requires both Zn2+ (for substrate polarisation and transition-state stabilisation) and cysteine residues (which coordinate the zinc and participate in catalysis).

Calculate enzyme activity from your data – includes Beer-Lambert conversion and common-mistake warnings:

Pyruvate Kinase Coupled Assay (IBO 2017)

Pyruvate kinase catalyses:

Neither substrate nor product absorbs distinctively, so the assay is coupled to lactate dehydrogenase (LDH), which is added in large excess:

The decrease in A340 is stoichiometric with PEP consumption, because excess LDH ensures that the LDH step is never rate-limiting - the observed rate reflects only PK activity.

The 2017 exam embedded this kinetics work in a clinical context: a family with suspected pyruvate kinase deficiency (PKD), the most common cause of hereditary non-spherocytic haemolytic anaemia. Students calculated haematological indices (MCV, MCH, MCHC) from blood data, determined PK kinetic parameters, and connected the biochemistry to a pedigree analysis.

Why PKD causes anaemia: mature red blood cells lack mitochondria and depend entirely on glycolysis for ATP. PK is the final, irreversible step of glycolysis. Reduced PK activity means reduced ATP, which means RBCs cannot maintain their membrane integrity and are destroyed prematurely (haemolysis).

Diaphorase Assay (IBO 2023)

Diaphorase is a flavoprotein (contains FAD and iron-sulfur centres, MW ~23 kDa) that catalyses:

The reduced NBT formazan absorbs at 595 nm - the same wavelength as the Bradford assay, which is a coincidence but can cause confusion if you mix up your plates.

The 2023 exam combined Ni-NTA purification, Bradford quantification, and diaphorase kinetics into a single integrated practical, culminating in $V_{\max}$ and $k_{\text{cat}}$ calculations.

Worked example from the 2023 official key:

Given: at [S] = 20 uM, observed activity = 6500 umol min$^{-1}$ mg$^{-1}$; $K_M$ = 10 uM.

Plot your own kinetics data – enter [S] and v0 values, fit the Michaelis-Menten curve, and toggle a Hanes-Woolf linearisation:

SDS-PAGE

Why it works (first principles)

SDS (sodium dodecyl sulfate) is a detergent that denatures proteins and coats them with negative charge proportional to their length. This means all SDS-coated proteins have roughly the same charge-to-mass ratio, so separation in the polyacrylamide gel is purely by size: smaller proteins migrate faster.

After electrophoresis, the gel is stained (usually Coomassie Blue) so bands become visible. Molecular weight is estimated by comparing the migration distance (Rf = distance protein / distance dye front) to a standard ladder.

SDS-PAGE appeared in the 2016 IBO exam as part of a protein expression and purification workflow, where students had to:

• Calculate loading volumes to achieve a target cell concentration per lane
• Load samples in the correct order
• Estimate molecular weights from the gel using a log(MW) vs Rf calibration plot
• Interpret gel-filtration chromatography data to determine whether the purified proteins form homo- or hetero-oligomers

Exam traps

• Incorrect loading-volume calculations (forgetting dilution factors).
• Damaging the gel while opening the cassette.
• Plotting Rf incorrectly.
• Confusing gel-filtration retention time (larger = earlier elution) with molecular weight.

Antioxidant Assays (DPPH)

The 2016 exam included a DPPH radical-scavenging assay. DPPH is a stable free radical with a deep purple colour ($\lambda_{\max}$ = 517 nm). When an antioxidant donates an electron or hydrogen atom, DPPH is reduced to colourless DPPH-H, and the absorbance drops.

The key metric is SC50: the concentration of antioxidant that scavenges 50% of the DPPH radicals. Lower SC50 = stronger antioxidant.

Plot SC% vs log(concentration), interpolate to find the concentration at SC% = 50.

Exam trap: a very common mistake is claiming that a higher SC50 means a stronger antioxidant - it is the opposite.

Fermentation Kinetics

Also from the 2016 exam: a homolactic Lactobacillus strain is grown in MRS medium. Growth is tracked by OD600, and lactic acid production is quantified by acid-base titration with 0.1 M NaOH.

Key formulas:

Generation time during exponential growth:

Lactic acid from titration (1 mol lactic acid reacts with 1 mol NaOH; MW lactic acid = 90 g/mol, MW NaOH = 40 g/mol):

Exam traps: uncalibrated pH electrode, stir bar striking the electrode, endpoint missed (pH outside 6.95-7.05), forgetting dilution factor.

Haematological Indices

The 2017 exam required calculation and interpretation of blood indices. These formulas come up whenever the practical has a clinical-case component.

Classification:

In PKD, the typical pattern is normocytic/normochromic anaemia with elevated reticulocytes (the bone marrow compensating by releasing immature RBCs).

Compute blood indices from CBC data – includes IBO 2017 exam presets and automatic anaemia classification:

Chromatography and Electrophoresis Theory

The 2023 exam included True/False questions about ion-exchange and size-exclusion chromatography that required conceptual understanding, not just recall.

Ion-exchange chromatography: proteins bind based on net charge, which depends on the relationship between the protein’s isoelectric point (pI) and the buffer pH. At pH > pI, the protein is negatively charged and binds an anion exchanger (like DEAE-Sepharose). At pH < pI, the protein is positively charged and binds a cation exchanger. Elution is by increasing salt (ionic strength competition) or changing pH.

Size-exclusion (gel-filtration) chromatography: smaller proteins enter the pores of the beads and take a longer path through the column, so they elute later. Larger proteins (or complexes) are excluded from the pores and elute earlier. A calibration curve of log(MW) vs elution volume allows estimation of the native molecular weight and hence the oligomeric state.

Key principle: in gel filtration, larger = earlier elution. In SDS-PAGE, larger = slower migration. These are opposite directions and easy to confuse.

E. coli Expression Strain Selection

The 2023 exam tested knowledge of specialised E. coli strains for recombinant protein expression: