Immobilized pH gradient (IPG) methods MCQs With Answer

Introduction: Immobilized pH gradient (IPG) methods are a cornerstone of high-resolution protein separation and characterization in pharmaceutical research. This blog of IPG MCQs with answers is tailored for M.Pharm students to deepen understanding of IPG theory, strip chemistry, sample preparation, focusing conditions, gradient selection and troubleshooting. Questions emphasize practical aspects—immobiline chemistry, rehydration protocols, effects of denaturants and reducing agents, narrow vs broad gradients, and integration with SDS-PAGE for 2D electrophoresis. The set challenges conceptual knowledge and formulation-related impacts on protein stability and migration, preparing students for lab practice, exams, and formulation design decisions involving IEF techniques.

Q1. What is the defining feature of an immobilized pH gradient (IPG) strip used in isoelectric focusing?

• pH gradient formed by ampholytes that migrate during focusing
• Covalently linked buffering groups within the polyacrylamide matrix creating a fixed pH gradient
• Gradient produced by an external pH reservoir applied to strip ends
• pH gradient generated by temperature differences along the strip

Correct Answer: Covalently linked buffering groups within the polyacrylamide matrix creating a fixed pH gradient

Q2. Which chemical class is used to create the immobilized buffering groups in IPG gels?

• Carrier ampholytes
• Immobilines (acrylamide derivatives with titratable groups)
• Polyethylene glycol
• Non-ionic detergents

Correct Answer: Immobilines (acrylamide derivatives with titratable groups)

Q3. Compared to carrier ampholyte-based IEF, a primary advantage of IPG strips is:

• Greater susceptibility to cathodic drift
• Higher reproducibility and stable, immobilized pH profile
• Requirement for continuous addition of ampholytes during run
• Lower resolution for closely spaced pI proteins

Correct Answer: Higher reproducibility and stable, immobilized pH profile

Q4. Which component is commonly included in IPG strip rehydration buffers to maintain proteins in a denatured but soluble state?

• High concentration of sodium chloride (NaCl)
• Urea and thiourea with non-ionic or zwitterionic detergents
• Strong acids to lower pH
• Organic solvents like methanol as primary denaturant

Correct Answer: Urea and thiourea with non-ionic or zwitterionic detergents

Q5. Why are reducing agents such as dithiothreitol (DTT) used during IPG rehydration/focusing?

• To fix proteins covalently in the gel
• To prevent carbamylation of lysines
• To reduce disulfide bonds and keep proteins in a reduced, unfolded state
• To increase ionic strength for better focusing

Correct Answer: To reduce disulfide bonds and keep proteins in a reduced, unfolded state

Q6. Which of the following is a common problem in IPG focusing and is mitigated by including ampholytes or detergents in the sample buffer?

• Over-polymerization of the gel
• Protein precipitation/aggregation causing poor focusing
• Excessive pH immobilization
• Increased photobleaching of proteins

Correct Answer: Protein precipitation/aggregation causing poor focusing

Q7. Narrow-range IPG strips (e.g., pH 4–7) are preferred when:

• Analyzing crude cell lysates with highly diverse pI distribution
• High-resolution separation of proteins within a limited pI window is required
• One desires to reduce resolution for faster runs
• Quantifying total protein concentration regardless of pI

Correct Answer: High-resolution separation of proteins within a limited pI window is required

Q8. Which factor most directly determines the position where a protein focuses in an IPG strip?

• Molecular weight of the protein
• Net charge at local pH (isoelectric point, pI) relative to the gradient
• Concentration of acrylamide in the gel
• Volume of rehydration buffer used

Correct Answer: Net charge at local pH (isoelectric point, pI) relative to the gradient

Q9. What is the rationale for equilibration of an IPG strip in SDS-containing buffer before the second-dimension SDS-PAGE?

• To re-establish the IPG pH gradient for transfer
• To introduce SDS that coats proteins, imparting uniform negative charge for SDS-PAGE separation by size
• To remove immobilines from the strip
• To dehydrate the strip for storage

Correct Answer: To introduce SDS that coats proteins, imparting uniform negative charge for SDS-PAGE separation by size

Q10. Which of the following conditions increases risk of protein carbamylation during IPG sample preparation?

• Using freshly prepared urea solutions at room temperature for short times
• Heating urea solutions or long storage leading to decomposition to cyanate
• Adding 10% glycerol to rehydration buffer
• Performing focusing at low voltages

Correct Answer: Heating urea solutions or long storage leading to decomposition to cyanate

Q11. Immobilized pH gradients are created during gel casting by mixing monomers with different:

• Polymerization initiators only
• Immobiline ratios (acidic and basic immobilines) to set local buffering capacity
• Salt concentrations applied at strip ends
• Temperature gradients across the casting mold

Correct Answer: Immobiline ratios (acidic and basic immobilines) to set local buffering capacity

Q12. Which practice reduces focusing artifacts such as horizontal streaking in IPG-based 2D gels?

• Omitting reducing agents during rehydration
• Using appropriate detergents, removing salts, and ensuring complete solubilization of proteins
• Increasing protein load without changing buffer composition
• Using extremely high acrylamide percentage in IPG strips

Correct Answer: Using appropriate detergents, removing salts, and ensuring complete solubilization of proteins

Q13. What is the typical purpose of adding ampholytes to IPG strip rehydration solution even though the strip contains immobilized buffering groups?

• They are required to generate the pH gradient in IPG strips
• To act as carrier molecules improving solubilization and resolution of very basic/acidic proteins and preventing sample overload artifacts
• To polymerize the gel during storage
• To fix proteins irreversibly to the gel matrix

Correct Answer: To act as carrier molecules improving solubilization and resolution of very basic/acidic proteins and preventing sample overload artifacts

Q14. When designing IPG experiments for membrane proteins, which modification to sample preparation is most beneficial?

• Exclude all detergents to avoid interference
• Include zwitterionic detergents and thiourea to improve solubilization while preserving IEF compatibility
• Use only SDS during IEF for best focusing
• Lower urea concentration to avoid denaturation

Correct Answer: Include zwitterionic detergents and thiourea to improve solubilization while preserving IEF compatibility

Q15. What is “cathodic drift” in IPG focusing and how is it typically controlled?

• A drift of proteins toward the anode due to contamination; controlled by adding more reducing agent
• Progressive migration of the entire pH gradient toward the cathode due to electroosmotic flow; minimized by optimized strip chemistry and run conditions
• Sudden collapse of gel polymerization; controlled by lowering voltage
• Evaporation of rehydration buffer causing shrinkage; controlled by sealing the strip

Correct Answer: Progressive migration of the entire pH gradient toward the cathode due to electroosmotic flow; minimized by optimized strip chemistry and run conditions

Q16. Which parameter is most critical when selecting IPG strip pH range for analyzing isoforms of a therapeutic protein with known pI 6.2?

• Choose a broad pH 3–10 to cover all possibilities
• Select a narrow-range strip centered around pH 5.5–7.0 to increase resolution near pI 6.2
• Use an alkaline strip only (pH 8–11)
• pH range is irrelevant if protein is reduced

Correct Answer: Select a narrow-range strip centered around pH 5.5–7.0 to increase resolution near pI 6.2

Q17. In IPG strip rehydration, passive in-gel rehydration vs active rehydration (electro-rehydration): the main advantage of passive rehydration is:

• Much faster rehydration times
• Lower risk of sample displacement and simpler, reproducible loading for many preparations
• Ability to focus while rehydrating
• Higher final protein concentration in focused bands

Correct Answer: Lower risk of sample displacement and simpler, reproducible loading for many preparations

Q18. Which of the following best explains why IPG strips improve inter-run reproducibility compared to carrier ampholyte IEF?

• IPG strips use higher voltages only
• The covalently immobilized buffering groups form a fixed gradient that does not vary between runs
• Carrier ampholytes are more stable than immobilines
• IPG strips require no equilibration step before SDS-PAGE

Correct Answer: The covalently immobilized buffering groups form a fixed gradient that does not vary between runs

Q19. Which storage condition is recommended for dry IPG strips to maintain performance before use?

• Room temperature, exposed to air
• Frozen at -80°C without sealing
• Sealed and stored at recommended cool temperatures (e.g., 4°C) and protected from moisture
• Immersed in strong acid solution for stability

Correct Answer: Sealed and stored at recommended cool temperatures (e.g., 4°C) and protected from moisture

Q20. During troubleshooting, a student notices increased streaking and loss of spots when loading high protein amounts onto an IPG strip. The best immediate remedial action is:

• Increase urea concentration to 12 M
• Reduce protein load, remove salts, and optimize detergents to improve solubility and reduce overloading artifacts
• Skip equilibration steps and run directly to SDS-PAGE
• Dry the strip before focusing to concentrate proteins

Correct Answer: Reduce protein load, remove salts, and optimize detergents to improve solubility and reduce overloading artifacts

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