Isoelectric focusing MCQs With Answer

Isoelectric focusing (IEF) MCQs With Answers for M. Pharm Students

Isoelectric focusing is a high-resolution electrophoretic technique that separates amphoteric molecules—especially proteins and peptides—solely based on their isoelectric points (pI) within a stable pH gradient. It is foundational to modern pharmaceutical analytical techniques for characterizing biopharmaceuticals, mapping charge variants, resolving isoforms, and preparing samples for 2D electrophoresis and mass spectrometry. This quiz is designed to reinforce conceptual clarity on gradient chemistry (carrier ampholytes vs IPG), operational parameters (voltage, salts, temperature), sample preparation (urea, CHAPS, DTT), and detection strategies. Each MCQ targets practical understanding and troubleshooting skills so you can confidently apply IEF to protein profiling, comparability studies, and critical quality attribute assessment in pharmaceutical analysis.

Q1. What is the fundamental principle behind isoelectric focusing (IEF) separation?

• Molecules migrate in a pH gradient until their net charge becomes zero at their pI
• Molecules are separated purely by size exclusion through gel pores
• Molecules bind selectively to immobilized ligands of complementary affinity
• Molecules partition based on hydrophobicity between two phases

Correct Answer: Molecules migrate in a pH gradient until their net charge becomes zero at their pI

Q2. What is the primary role of carrier ampholytes in IEF?

• Generate and buffer a stable pH gradient under an electric field
• Covalently immobilize the pH gradient within the gel matrix
• Provide a reducing environment to prevent disulfide formation
• Visualize proteins after focusing

Correct Answer: Generate and buffer a stable pH gradient under an electric field

Q3. Which statement correctly describes protein behavior relative to its pI in IEF?

• At pH > pI, a protein is negatively charged and migrates toward the anode
• At pH > pI, a protein is positively charged and migrates toward the cathode
• At pH < pI, a protein is negatively charged and migrates toward the anode
• A protein’s charge is zero at any pH when an electric field is applied

Correct Answer: At pH > pI, a protein is negatively charged and migrates toward the anode

Q4. How is an immobilized pH gradient (IPG) created in IPG gels?

• By covalently incorporating weak acid/base “Immobiline” monomers into the polyacrylamide matrix
• By recycling carrier ampholytes to maintain dynamic buffering capacity
• By overlaying agarose layers of different pH values
• By adding strong acids and bases during the run

Correct Answer: By covalently incorporating weak acid/base “Immobiline” monomers into the polyacrylamide matrix

Q5. How does choosing a narrower pH range (e.g., 4–5) affect IEF resolution?

• It increases resolution by steepening the pH gradient per unit distance
• It decreases resolution by limiting protein mobility
• It has no effect on resolution, only on run time
• It causes cathodic drift and increased band broadening

Correct Answer: It increases resolution by steepening the pH gradient per unit distance

Q6. Which sample buffer composition is recommended for protein solubilization in IEF?

• Urea with zwitterionic detergents (e.g., CHAPS); avoid SDS during focusing
• Replace urea with SDS to enhance charge-based focusing
• Use only ionic detergents to sharpen protein bands
• No detergents or chaotropes should be present during IEF

Correct Answer: Urea with zwitterionic detergents (e.g., CHAPS); avoid SDS during focusing

Q7. Which gel matrix is most commonly used for IEF in modern proteomics workflows?

• Polyacrylamide gels (including IPG strips)
• Cellulose acetate membranes
• Agarose gels exclusively
• Silica-based thin-layer plates

Correct Answer: Polyacrylamide gels (including IPG strips)

Q8. Which factor most commonly causes band broadening and poor focusing in IEF?

• Excessive salt in the sample leading to high conductivity and Joule heating
• Use of narrow pH range IPG strips
• Low ampholyte concentration in IPG runs
• Operating at controlled, low temperature

Correct Answer: Excessive salt in the sample leading to high conductivity and Joule heating

Q9. Which physicochemical property of a protein determines its final focusing position in IEF?

• Molecular mass
• Hydrophobicity
• Isoelectric point (pI)
• Concentration

Correct Answer: Isoelectric point (pI)

Q10. In two-dimensional electrophoresis (2-DE), what follows IEF as the second dimension?

• SDS-PAGE separation by molecular mass
• Native PAGE separation by shape
• Capillary electrophoresis separation by charge
• Gel filtration separation by size

Correct Answer: SDS-PAGE separation by molecular mass

Q11. What is a key advantage of IPG over carrier ampholyte IEF?

• Highly reproducible, stable pH gradients with minimal cathodic drift
• Lower resolution due to fixed buffer capacity
• Ability to adjust gradient slope during the run
• Elimination of any need for temperature control

Correct Answer: Highly reproducible, stable pH gradients with minimal cathodic drift

Q12. Which electrode solutions are typically used in carrier ampholyte slab IEF?

• Anode: acidic solution (e.g., phosphoric acid); Cathode: basic solution (e.g., NaOH)
• Anode: NaOH; Cathode: phosphoric acid
• Both electrodes: neutral phosphate buffer
• No electrode solutions are used in IEF

Correct Answer: Anode: acidic solution (e.g., phosphoric acid); Cathode: basic solution (e.g., NaOH)

Q13. How does increasing the applied voltage primarily influence an IEF run (within thermal limits)?

• It accelerates focusing and can narrow bands without changing the pI position
• It permanently shifts the pI of analytes to higher values
• It converts IEF into size-based separation
• It eliminates the need for ampholytes or IPGs

Correct Answer: It accelerates focusing and can narrow bands without changing the pI position

Q14. What is the main function of urea in IEF sample preparation?

• To denature and solubilize proteins without imparting strong ionic charge
• To provide the pH gradient through buffering action
• To covalently fix proteins at their pI
• To act as a high-conductivity salt for faster runs

Correct Answer: To denature and solubilize proteins without imparting strong ionic charge

Q15. Which staining method is generally the most sensitive for protein detection after IEF?

• Silver staining
• Coomassie Brilliant Blue
• Ponceau S
• Amido Black

Correct Answer: Silver staining

Q16. Which detection approach is commonly used in capillary IEF (cIEF) for on-column monitoring?

• Whole-column UV absorbance detection
• Flame ionization detection
• Evaporative light scattering detection
• Refractive index detection

Correct Answer: Whole-column UV absorbance detection

Q17. How can carbamylation artifacts from urea be minimized during IEF sample prep and running?

• Use fresh, deionized urea and maintain lower temperatures during focusing
• Run at elevated temperatures to increase ion mobility
• Add SDS to mask carbamylation
• Use strong base to hydrolyze all carbamylated residues

Correct Answer: Use fresh, deionized urea and maintain lower temperatures during focusing

Q18. How does increased sialylation (e.g., in glycoprotein charge variants) typically affect pI?

• Decreases pI due to additional acidic moieties
• Increases pI due to removal of negative charges
• Does not change pI but alters molecular mass only
• Eliminates focusing by neutralizing charge

Correct Answer: Decreases pI due to additional acidic moieties

Q19. Why are reducing agents (e.g., DTT) included in IEF sample buffers?

• To cleave disulfide bonds, improve solubility, and reduce streaking
• To generate the pH gradient more rapidly
• To covalently fix proteins at the focused position
• To increase the net negative charge of proteins

Correct Answer: To cleave disulfide bonds, improve solubility, and reduce streaking

Q20. You need to resolve protein isoforms differing by ~0.02 pH units. Which single change most effectively improves resolution?

• Use a very narrow-range IPG strip (e.g., pH 4.5–5.5) that brackets the isoforms
• Increase sample load fivefold without other changes
• Widen the pH range to 3–10
• Remove ampholytes to reduce background

Correct Answer: Use a very narrow-range IPG strip (e.g., pH 4.5–5.5) that brackets the isoforms

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