Resolution and reproducibility in 2D gels MCQs With Answer

Resolution and reproducibility in 2D gels MCQs With Answer

Introduction: Two-dimensional (2D) gel electrophoresis remains a cornerstone technique in protein analysis for separating complex mixtures by isoelectric point and molecular weight. For M.Pharm students working on proteins and formulations, understanding resolution — the ability to distinguish closely related protein species — and reproducibility — the capacity to obtain consistent results across runs — is essential. This quiz package focuses on practical and theoretical aspects that influence spot sharpness, spot capacity, gel chemistry, sample preparation, detection methods, and data normalization. The questions emphasize factors to optimize separation and ensure reliable comparative proteomics, troubleshooting artifacts, and best practices for producing reproducible, high-resolution 2D gels.

Q1. What best defines resolution in 2D gel electrophoresis?

• Ability to separate proteins that differ slightly in isoelectric point and molecular weight
• Capacity to load the maximum possible protein amount on a gel
• Speed at which an IEF run completes
• Sensitivity of the staining method used to visualize proteins

Correct Answer: Ability to separate proteins that differ slightly in isoelectric point and molecular weight

Q2. What is the primary advantage of using immobilized pH gradient (IPG) strips for the first dimension?

• They are cheaper than carrier ampholytes
• They allow reproducible, stable pH gradients for isoelectric focusing
• They prevent proteins from entering the second dimension
• They eliminate the need for SDS in the second dimension

Correct Answer: They allow reproducible, stable pH gradients for isoelectric focusing

Q3. Which practice most improves reproducibility between 2D gels?

• Varying acrylamide concentration for each run
• Using internal standards and identical IPG strip rehydration and focusing conditions
• Running gels at different voltages each time to optimize speed
• Pooling different sample types on each gel

Correct Answer: Using internal standards and identical IPG strip rehydration and focusing conditions

Q4. What is the most common effect of sample overloading on 2D gel resolution?

• Sharper, well-defined spots for low-abundance proteins
• Horizontal and vertical streaking with spot fusion and loss of resolution
• Complete disappearance of all protein spots
• Improved detection of post-translational modifications

Correct Answer: Horizontal and vertical streaking with spot fusion and loss of resolution

Q5. Why is equilibration between the first (IEF) and second (SDS-PAGE) dimensions necessary?

• To dehydrate the IPG strip before SDS-PAGE
• To reduce disulfide bonds, alkylate cysteines and saturate proteins with SDS for uniform charge-to-mass ratios
• To increase the pH of the IPG strip to basic conditions
• To remove low molecular weight contaminants that moved in IEF

Correct Answer: To reduce disulfide bonds, alkylate cysteines and saturate proteins with SDS for uniform charge-to-mass ratios

Q6. How does acrylamide concentration in the second-dimension gel affect protein separation?

• It controls pore size and thus the molecular weight range resolved
• It changes the isoelectric points of proteins
• It only affects staining intensity, not separation
• It determines the pH gradient of the first dimension

Correct Answer: It controls pore size and thus the molecular weight range resolved

Q7. Compared to carrier ampholytes, carrier-free IPG strips typically provide:

• Less reproducible pH gradients but higher sensitivity
• Highly reproducible, stable pH gradients and better spot position reproducibility
• No separation by isoelectric point
• Faster SDS-PAGE run times

Correct Answer: Highly reproducible, stable pH gradients and better spot position reproducibility

Q8. Which staining method gives high sensitivity while remaining compatible with downstream mass spectrometry?

• Classical silver staining with formaldehyde developer
• Coomassie Brilliant Blue R-250
• Fluorescent stains such as SYPRO Ruby
• Ruthenium-based fluorescent stains that are incompatible with MS

Correct Answer: Fluorescent stains such as SYPRO Ruby

Q9. Vertical streaking on the second-dimension gel is most often caused by:

• High-salt contamination only
• Protein aggregation or incomplete reduction/alkylation leading to SDS-insufficient denaturation
• Use of IPG strips instead of carrier ampholytes
• Excessive gel polymerization time

Correct Answer: Protein aggregation or incomplete reduction/alkylation leading to SDS-insufficient denaturation

Q10. Which quantitative metric is commonly used to assess reproducibility of spot intensity across replicate gels?

• Signal-to-noise ratio of a single spot
• Coefficient of variation (CV) of spot volumes or intensities
• Absolute migration distance of the gel front
• Number of spots detected on one gel only

Correct Answer: Coefficient of variation (CV) of spot volumes or intensities

Q11. Alkylation of cysteine residues after reduction is performed to:

• Enhance staining intensity of basic proteins
• Prevent reformation of disulfide bonds and stabilize proteins for SDS-PAGE
• Raise the isoelectric point of proteins
• Promote protein aggregation to increase spot size

Correct Answer: Prevent reformation of disulfide bonds and stabilize proteins for SDS-PAGE

Q12. What does the term “dynamic range” refer to in the context of 2D gel proteomics?

• The range of gel voltages used during IEF
• The difference in abundance between the most and least abundant proteins detected on a gel
• The number of pH units covered by an IPG strip
• The dynamic movement of proteins during equilibration

Correct Answer: The difference in abundance between the most and least abundant proteins detected on a gel

Q13. Which approach improves automated spot matching across different gels during image analysis?

• Use of single-point manual alignment only
• Use of internal landmark proteins/standards and computational warping algorithms
• Random cropping of gel images before analysis
• Varying staining protocols between gels to generate unique patterns

Correct Answer: Use of internal landmark proteins/standards and computational warping algorithms

Q14. How does narrowing the pH range of the first-dimension IPG strip (e.g., pH 4–7 instead of 3–10) affect resolution?

• Decreases resolution because fewer proteins focus
• Increases resolution within that pH window, allowing separation of proteins with small pI differences
• Eliminates the need for equilibration steps
• Only affects staining but not separation

Correct Answer: Increases resolution within that pH window, allowing separation of proteins with small pI differences

Q15. Typical sample amounts for analytical versus preparative 2D gels are:

• Analytical: 10–100 µg; Preparative: several hundred micrograms to milligrams
• Analytical: 1–5 mg; Preparative: 10–20 µg
• Both use identical amounts: always 50 µg
• Analytical: nanogram range; Preparative: picogram range

Correct Answer: Analytical: 10–100 µg; Preparative: several hundred micrograms to milligrams

Q16. What is the role of zwitterionic detergents (e.g., CHAPS) in 2D gel sample buffers?

• They change the isoelectric point of proteins during IEF
• They solubilize proteins, including membrane proteins, without interfering with IEF
• They polymerize the acrylamide gel
• They act as reducing agents for disulfide bonds

Correct Answer: They solubilize proteins, including membrane proteins, without interfering with IEF

Q17. Best storage conditions for unused dry IPG strips to maintain reproducibility are:

• Store dry, sealed in moisture-proof pouches, protected from light at recommended temperatures (room temperature or 4°C)
• Keep rehydrated strips frozen indefinitely
• Expose strips to ambient humidity to maintain flexibility
• Store strips in direct sunlight to maintain pH gradient stability

Correct Answer: Store dry, sealed in moisture-proof pouches, protected from light at recommended temperatures (room temperature or 4°C)

Q18. Which modification commonly causes a shift in isoelectric point (pI) observed as a spot train or spot shift on a 2D gel?

• Proteolytic cleavage that doesn’t change charge
• Phosphorylation adding negative charges and shifting pI toward acidic values
• Glycosylation that only affects molecular weight but not pI
• Dialysis against neutral buffer

Correct Answer: Phosphorylation adding negative charges and shifting pI toward acidic values

Q19. Which normalization strategy is widely accepted for quantitative comparison of spot intensities between gels?

• Normalization to the running time of the gel
• Normalization to total spot volume or to internal standard (housekeeping) spots
• Normalization by increasing staining time arbitrarily
• Normalization by cropping the gel image randomly

Correct Answer: Normalization to total spot volume or to internal standard (housekeeping) spots

Q20. Why is reproducibility critical in 2D gel–based comparative proteomics studies?

• Because reproducible gels allow reliable detection of biologically meaningful differences and statistical analysis across replicates
• Because reproducibility increases the dye binding of proteins
• Because reproducibility eliminates the need for mass spectrometry confirmation
• Because reproducible gels run faster than non-reproducible ones

Correct Answer: Because reproducible gels allow reliable detection of biologically meaningful differences and statistical analysis across replicates

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