Question 1

Principle of ion-exchange chromatography: In this protein purification method, separation primarily occurs on the basis of which property?

Accepted Answer

Introduction / Context: Ion-exchange chromatography is a staple of protein purification. Resins bearing fixed charges bind oppositely charged protein surfaces, allowing selective retention and elution. Given Data / Assumptions: Stationary phase carries negative (cation exchanger) or positive (anion exchanger) charges. Mobile phase pH and ionic strength are controlled. Proteins possess pH-dependent net charges (related to pI). Concept / Approach: Binding affinity depends on the protein’s net charge at the working pH. Proteins with stronger opposite net charge bind tighter; elution is achieved by salt gradients or pH shifts that weaken electrostatic interactions. Step-by-Step Solution: Select exchanger type (for example, DEAE for anion exchange).Set pH so the target has appropriate charge.Elute by increasing salt or changing pH, separating proteins by net charge. Verification / Alternative check: Elution order typically correlates with isoelectric points: proteins further from the mobile phase pH (thus larger net charge magnitude) elute later. Why Other Options Are Wrong: Molecular size / shape: These govern gel filtration, not ion exchange. Hydrophobicity: Basis of hydrophobic interaction chromatography. Isoelectric precipitation: A separate technique, not the ion-exchange mechanism. Common Pitfalls: Running near the protein’s pI reduces binding; mismatch of exchanger type leads to poor resolution. Final Answer: Net charge on the protein.

Question 2

Stabilizing proteins after yeast cell disruption: Which additive(s) can be included to protect protein structure and activity immediately after mechanical lysis?

Accepted Answer

Introduction / Context: Upon disrupting yeast or other cells, endogenous proteases, shear stress, and ionic imbalances threaten protein integrity. Appropriate additives can stabilize targets and improve downstream purification yield. Given Data / Assumptions: Lysis releases proteases and nucleases. Protein aggregation is promoted by inappropriate salt or pH. Some enzymes require ligands (for example, nucleotides) for stability. Concept / Approach: Adding salt such as NaCl tunes ionic strength, minimizing nonspecific electrostatic interactions. Protease inhibitors (for example, PMSF, leupeptin cocktails) suppress proteolysis. Ligands like AMP or cofactors can stabilize the native conformation of allosteric or nucleotide-binding proteins, reducing denaturation and loss of activity. Step-by-Step Solution: Supplement lysis buffer with NaCl (commonly 100–300 mM).Add a broad-spectrum protease inhibitor mix at recommended concentration.Include stabilizing ligands (AMP, ATP analogs, metal ions) if compatible with the target. Verification / Alternative check: Compare activity recoveries with and without additives; improved activity and reduced degradation bands on SDS-PAGE confirm protection. Why Other Options Are Wrong: Single additive only: Often insufficient; multiple stressors exist after lysis. Detergent only: Useful for membranes but does not address proteolysis or charge screening alone. Common Pitfalls: Forgetting EDTA/EGTA when metalloproteases are present; adding ligands that interfere with downstream binding (for example, affinity resins). Final Answer: All of these.

Question 3

Affinity chromatography fundamentals: What is the core principle by which affinity chromatography separates biomolecules?

Accepted Answer

Introduction / Context: Affinity chromatography exploits natural or engineered high-affinity interactions (for example, enzyme–substrate analog, antibody–antigen, His-tag–Ni2+) to purify a specific protein from complex mixtures with high selectivity. Given Data / Assumptions: A stationary phase presents immobilized ligand (for example, Ni-NTA, Protein A, biotin, substrate analog). Target protein possesses a complementary binding site or tag. Elution occurs by competitive ligand, pH, or ionic strength change. Concept / Approach: Only biomolecules with the correct binding epitope are retained strongly. Non-specific binders are washed away under conditions that preserve the specific interaction. Elution disrupts the specific interaction while maintaining protein integrity. Step-by-Step Solution: Bind: load clarified lysate onto ligand-bearing resin.Wash: remove weak/non-specific binders.Elute: add competitor or change pH/salt to release target. Verification / Alternative check: Single major band by SDS-PAGE after elution indicates specificity; activity assays confirm functional recovery. Why Other Options Are Wrong: Aggregation/precipitation: Not the mechanism of affinity capture. Random silica adsorption/hydrophobic forces: Describe other chromatographies (HIC, reversed-phase), not affinity. Gas–liquid partitioning: Pertains to GC, irrelevant to proteins in water. Common Pitfalls: Overloading columns, using incompatible buffers that block binding (for example, imidazole during Ni-NTA loading). Final Answer: Specific binding of a protein to an immobilized ligand (high-affinity interaction).

Question 4

Calculating specific activity in enzyme purification: A purified sample contains 10 μg of protein and an enzyme activity of 1 unit (1 μmol ATP synthesized per second). What is the specific activity of this final sample?

Accepted Answer

Introduction / Context: Specific activity is a central metric in enzyme purification, defined as catalytic units per milligram of total protein. It summarizes both purity and functional yield across steps. Given Data / Assumptions: Total protein in the sample: 10 μg. Total activity: 1 unit (1 μmol product per second). Units are per second; mass must be expressed in mg. Concept / Approach: Specific activity = total activity / mass of protein. Convert micrograms to milligrams before division to keep units consistent. Step-by-Step Solution: Convert mass: 10 μg = 10 * 10^-6 g = 0.01 mg.Compute: specific activity = 1 unit / 0.01 mg.Result: 100 units/mg. Verification / Alternative check: Check dimensions: (units) / (mg) gives units/mg. If the same activity were in 1 mg, value would be 1 unit/mg; because mass is 100× smaller (0.01 mg), specific activity is 100× larger (100 units/mg). Why Other Options Are Wrong: 50 or 0.1 units/mg: Arise from incorrect mass conversion or arithmetic. 1,000 or 10,000 units/mg: Overestimate by factors of 10–100. Common Pitfalls: Forgetting to convert μg to mg; confusing specific activity with total activity; neglecting to report standardized units. Final Answer: 100 units/mg.

Question 5

Trends during a successful protein purification scheme: Which changes do you typically expect as purification proceeds?

Accepted Answer

Introduction / Context: Protein purification aims to enrich the target relative to contaminants. Tracking total activity, total protein, specific activity, and yield at each step reveals progress and efficiency. Given Data / Assumptions: Specific activity = units per mg protein. Each step removes contaminants but may lose some target. Analytical checks include SDS-PAGE and activity assays. Concept / Approach: As impurities are removed, the denominator (total protein) drops faster than the numerator (activity) for a good protocol, so specific activity rises. Also, chromatography and fractionation reduce the number of proteins present. Total yield often declines somewhat due to inevitable losses. Step-by-Step Solution: Start with crude lysate: low specific activity, many proteins.Apply fractionation/columns: contaminants removed, target concentrated.Observe: specific activity up; band complexity on SDS-PAGE down. Verification / Alternative check: Purification tables show rising specific activity and decreasing total protein across steps, with yield tracked to ensure acceptable losses. Why Other Options Are Wrong: Specific activity decreases: Indicates poor or damaging procedures. Total yield always increases: Unrealistic; losses occur during handling. Common Pitfalls: Equating high yield with purity; over-optimizing for yield can sacrifice purity and vice versa. Final Answer: Both (a) and (c).

Question 6

Choosing separation strategies in protein purification: On which physicochemical properties can proteins be separated?

Accepted Answer

Introduction / Context: Successful purification usually combines orthogonal methods that exploit different properties of proteins. Recognizing available handles streamlines the design of a robust workflow. Given Data / Assumptions: Proteins vary in charge (pI), size, hydrophobicity, and solubility. Common techniques include ion exchange, salting-out, hydrophobic interaction, and size-exclusion chromatography. Target protein is stable under chosen conditions. Concept / Approach: Charge differences resolve proteins on ion exchangers. Solubility manipulations (for example, ammonium sulfate precipitation) concentrate proteins and remove contaminants. Size differences are exploited by gel filtration (SEC), which sieves molecules by hydrodynamic radius. Step-by-Step Solution: Screen conditions to find where target binds/precipitates appropriately.Sequence columns to maximize resolution with minimal denaturation.Monitor fractions by activity and SDS-PAGE. Verification / Alternative check: Orthogonality test: if two methods rank proteins differently, they complement each other and improve purity. Why Other Options Are Wrong: Only hydrophobicity: Overlooks many effective and widely used strategies. Common Pitfalls: Using methods that are not orthogonal, leading to marginal improvements; ignoring pH/pI alignment for ion exchange. Final Answer: All of these.

Question 7

Gel-filtration (size-exclusion) chromatography: What is the principal basis of separation when using porous beads packed in a column?

Accepted Answer

Introduction / Context: Gel-filtration (also called size-exclusion chromatography, SEC) separates macromolecules by size as they migrate through a matrix of porous beads. Given Data / Assumptions: Stationary phase: beads with a defined pore size distribution. Mobile phase: buffer that does not promote adsorption. Analytes: proteins of varying hydrodynamic radius. Concept / Approach: Larger molecules are excluded from more pores and thus traverse a shorter path, eluting earlier. Smaller molecules enter more pores, experience a longer residence time, and elute later. While molecular shape may subtly affect hydrodynamic radius, the governing parameter is effective size. Step-by-Step Solution: Pack column with appropriate pore-size beads.Load sample under non-adsorbing conditions.Collect fractions; earlier fractions contain larger species. Verification / Alternative check: Calibration with standards of known molecular mass/size yields a linear relationship between elution volume and log molecular mass over a defined range. Why Other Options Are Wrong: Shape only: Secondary effect; SEC is fundamentally size-based. Hydrophobicity/charge: Describe different chromatographies (HIC, ion exchange). Common Pitfalls: Using buffers that cause non-specific binding; overloading columns which degrades resolution. Final Answer: Size using porous beads packed in a column.

Question 8

Tracking a protein during purification: What is the most reliable way to locate your target enzyme in fractions collected from each step?

Accepted Answer

Introduction / Context: Purifying an enzyme requires a reliable readout to identify which fractions contain active protein. Activity assays directly report functional presence, making them superior to non-specific signals. Given Data / Assumptions: Target is an ATP-synthesizing enzyme (example used here). Fractions from steps such as precipitation or chromatography are available. Access to a simple, quantitative activity assay. Concept / Approach: While UV at 280 nm reveals total protein, it cannot distinguish the target from contaminants. Refractive index, conductivity, or broad mass spectrometry screens are non-specific or impractical at every step. A tailored activity assay is the gold standard for tracking the target and calculating specific activity and yield. Step-by-Step Solution: Establish an assay that measures ATP formation per unit time.Test each fraction; identify those with the highest specific activity.Pool active fractions and proceed to the next step. Verification / Alternative check: Correlate activity peaks with SDS-PAGE bands; specific activity should rise as purification advances. Why Other Options Are Wrong: UV 280 nm: Non-specific; many proteins absorb. Refractive index/conductivity: Reflect buffer composition, not enzyme identity. Mass spectrometry: Powerful but not routine for every fraction during process development. Common Pitfalls: Relying solely on A280 can mislead pooling decisions; always confirm with activity. Final Answer: Measure the rate of ATP synthesis (an activity assay specific to the target).

Protein Purification Questions