2D gel electrophoresis techniques MCQs With Answer

Introduction: 2D gel electrophoresis (2-DE) remains a cornerstone technique in proteomics and protein characterization, crucial for M.Pharm students focusing on proteins and formulations. This blog-style quiz collection of 20 MCQs with answers covers core concepts: isoelectric focusing (IEF) using IPG strips, SDS-PAGE in the second dimension, sample solubilization (urea/thiourea, detergents, reducing and alkylating agents), staining and visualization methods, DIGE, common artifacts, and compatibility with mass spectrometry. The questions are designed to reinforce practical understanding, troubleshooting strategies, and method optimization required in pharmaceutical protein analysis, making it a focused revision tool for advanced coursework and laboratory application.

Q1. Which property is primarily exploited by isoelectric focusing (first dimension) in 2D gel electrophoresis?

• Separation by isoelectric point (pI) where net charge becomes zero
• Separation by molecular weight due to sieving
• Separation by hydrophobicity using a nonpolar matrix
• Separation by enzymatic cleavage patterns

Correct Answer: Separation by isoelectric point (pI) where net charge becomes zero

Q2. What is the main advantage of using Immobilized pH Gradient (IPG) strips over carrier ampholyte-based IEF?

• IPG strips provide a stable, reproducible and immobilized pH gradient
• IPG strips eliminate the need for SDS in the second dimension
• IPG strips increase protein molecular weight resolution
• IPG strips remove all post-translational modifications

Correct Answer: IPG strips provide a stable, reproducible and immobilized pH gradient

Q3. Which combination of reagents is commonly used in 2D rehydration/sample buffer to solubilize proteins for IEF?

• 8 M urea, 2 M thiourea, 4% CHAPS, 20 mM DTT
• 1% SDS in water only
• 6 M guanidine hydrochloride without detergents
• Pure phosphate-buffered saline (PBS)

Correct Answer: 8 M urea, 2 M thiourea, 4% CHAPS, 20 mM DTT

Q4. What is the primary role of dithiothreitol (DTT) in 2D sample preparation?

• To reduce disulfide bonds to free thiols and maintain proteins in a reduced state
• To crosslink proteins and increase their molecular weight
• To oxidize cysteines forming additional disulfide bridges
• To act as a fluorescent dye for protein visualization

Correct Answer: To reduce disulfide bonds to free thiols and maintain proteins in a reduced state

Q5. Why is alkylation with iodoacetamide performed after reduction in 2D electrophoresis workflows?

• To block free thiols and prevent reformation of disulfide bonds
• To remove salts from the sample
• To stain proteins for visualization
• To increase protein hydrophobicity for better focusing

Correct Answer: To block free thiols and prevent reformation of disulfide bonds

Q6. The second dimension SDS-PAGE separates proteins primarily based on which characteristic?

• Apparent molecular weight due to SDS-induced uniform charge-to-mass ratio
• Intrinsic isoelectric point of the proteins
• Protein tertiary structure in native conformation
• Protein glycosylation status only

Correct Answer: Apparent molecular weight due to SDS-induced uniform charge-to-mass ratio

Q7. What is the purpose of the equilibration step between IEF and SDS-PAGE?

• To saturate the IPG strip with SDS and to perform reduction and alkylation for consistent denaturation
• To rehydrate the IPG strip with ampholytes for a second IEF run
• To induce protein renaturation before second dimension
• To cleave proteins into peptides for mass spectrometry

Correct Answer: To saturate the IPG strip with SDS and to perform reduction and alkylation for consistent denaturation

Q8. Which statement best describes the DIGE (Difference Gel Electrophoresis) approach?

• Proteins from different samples are labeled with spectrally distinct fluorescent dyes and co-run on the same 2D gel for direct quantitative comparison
• Proteins are digested in-gel and labeled after electrophoresis for identification
• IPG strips are dyed to reveal pH gradients before sample application
• Proteins are separated only by molecular weight using multiple SDS gels

Correct Answer: Proteins from different samples are labeled with spectrally distinct fluorescent dyes and co-run on the same 2D gel for direct quantitative comparison

Q9. Which staining method is traditionally considered most sensitive for detecting low-abundance protein spots on 2D gels?

• Silver staining
• Coomassie brilliant blue staining
• Amido black staining
• Methanol fixation without stain

Correct Answer: Silver staining

Q10. Horizontal streaking on 2D gels is often caused by which of the following issues?

• High salt concentration and poor protein solubilization
• Excessive gel polymerization time only
• Using too low a concentration of urea alone
• Applying too strong a reducing agent during staining

Correct Answer: High salt concentration and poor protein solubilization

Q11. In carrier ampholyte-based IEF, what is the primary function of ampholytes?

• To establish a continuous pH gradient across the gel
• To denature proteins prior to focusing
• To act as molecular weight markers
• To covalently bind proteins for immobilization

Correct Answer: To establish a continuous pH gradient across the gel

Q12. How is the isoelectric point (pI) of a protein defined?

• The pH at which the protein has no net electric charge
• The pH at which the protein is maximally soluble in water
• The pH at which the protein has its highest enzymatic activity
• The pH at which the protein binds most strongly to SDS

Correct Answer: The pH at which the protein has no net electric charge

Q13. Why is adding SDS during the isoelectric focusing step generally avoided?

• SDS imparts a uniform negative charge, disrupting separation by isoelectric point
• SDS increases protein hydrophobicity and enhances focusing too much
• SDS crosslinks proteins to the gel matrix making them immobile
• SDS fluoresces and interferes with staining methods

Correct Answer: SDS imparts a uniform negative charge, disrupting separation by isoelectric point

Q14. For improving detection of low-abundance proteins in 2D gels, which strategy is most appropriate?

• Prefractionation or enrichment (e.g., subcellular fractionation, affinity depletion) before 2D analysis
• Always increasing total sample load without any fractionation
• Omitting reducing agents to preserve native charge
• Running IEF at much lower voltages for shorter times

Correct Answer: Prefractionation or enrichment (e.g., subcellular fractionation, affinity depletion) before 2D analysis

Q15. What does passive rehydration of IPG strips typically involve?

• Soaking the strip overnight with rehydration buffer containing sample to allow proteins to enter the gel matrix
• Applying electric field during rehydration to force proteins into the strip
• Heating the strip at 95°C in water to accelerate uptake
• Vacuum-drying the strip with the sample on top

Correct Answer: Soaking the strip overnight with rehydration buffer containing sample to allow proteins to enter the gel matrix

Q16. Which staining approach is considered most compatible with subsequent mass spectrometry analysis?

• Colloidal Coomassie or fluorescent stains such as SYPRO Ruby
• Traditional silver staining with glutaraldehyde fixation
• Non-specific membrane staining without destaining
• Methylene blue staining in acidic solvents

Correct Answer: Colloidal Coomassie or fluorescent stains such as SYPRO Ruby

Q17. Why is thiourea often added together with urea in 2D buffers?

• Thiourea enhances solubilization of hydrophobic and membrane-associated proteins
• Thiourea acts as a polymerization catalyst for acrylamide
• Thiourea removes glycosylation from proteins
• Thiourea prevents protein staining during Coomassie staining

Correct Answer: Thiourea enhances solubilization of hydrophobic and membrane-associated proteins

Q18. What is a key reason to perform reduction and alkylation prior to IEF?

• To prevent disulfide bond reshuffling that causes horizontal streaking and spot distortion
• To increase the molecular weight of proteins for better SDS-PAGE separation
• To induce proteolytic cleavage for peptide mapping
• To enhance binding of Coomassie dye to proteins

Correct Answer: To prevent disulfide bond reshuffling that causes horizontal streaking and spot distortion

Q19. Which practice most improves 2D gel reproducibility between runs?

• Using IPG strips, standardized rehydration, voltage schedules and precise sample handling protocols
• Varying ampholyte concentrations randomly to test dynamic range
• Using different equilibration buffers for each run
• Changing gel acrylamide concentrations mid-run

Correct Answer: Using IPG strips, standardized rehydration, voltage schedules and precise sample handling protocols

Q20. Carbamylation of proteins is a known artifact in 2D electrophoresis. What causes carbamylation?

• Decomposition of urea to isocyanate at elevated temperature or pH, which reacts with amino groups
• Reaction of SDS with lysine residues during equilibration
• Excessive DTT causing alkylation of amines
• Exposure to silver stain reagents prior to electrophoresis

Correct Answer: Decomposition of urea to isocyanate at elevated temperature or pH, which reacts with amino groups

Download