Gel electrophoresis MCQs With Answer

Gel Electrophoresis MCQs With Answer is designed to help M. Pharm students master core analytical concepts used in research and pharmaceutical quality control. Gel electrophoresis underpins routine assays for nucleic acids and proteins—from agarose DNA analysis to SDS-PAGE protein profiling, isoelectric focusing, and capillary formats. Success in the lab requires understanding matrix selection, buffer chemistry, denaturation strategies (SDS, urea), visualization methods, and troubleshooting artifacts such as “smiling” and diffuse bands. These 20 MCQs probe principle, practice, and interpretation at an advanced level relevant to biopharmaceutical characterization, biosimilar comparability, and method development in modern pharmaceutical analytical techniques.

Q1. The fundamental principle of gel electrophoresis is best described as:

• Charged biomolecules migrate through a porous gel under an electric field and separate based on charge, size, and shape
• Molecules crystallize in a gel by reducing temperature
• Separation occurs due to solvent evaporation
• Molecules are separated by centrifugation speed only

Correct Answer: Charged biomolecules migrate through a porous gel under an electric field and separate based on charge, size, and shape

Q2. For resolving linear DNA fragments from about 0.5 kb to 20 kb, the most appropriate gel matrix is:

• Agarose gel electrophoresis
• Native polyacrylamide gel (PAGE)
• SDS-PAGE
• Capillary isoelectric focusing

Correct Answer: Agarose gel electrophoresis

Q3. In SDS-PAGE, the primary role of SDS is to:

• Denature proteins and impart a uniform negative charge proportional to mass
• Crosslink proteins to stabilize quaternary structure
• Reduce disulfide bonds
• Fluorescently label proteins

Correct Answer: Denature proteins and impart a uniform negative charge proportional to mass

Q4. In polyacrylamide gels, increasing %T (total acrylamide) will:

• Decrease pore size and improve resolution of smaller proteins
• Increase pore size and favor separation of large proteins
• Have no effect on pore size
• Only increase gel mechanical strength without affecting separation

Correct Answer: Decrease pore size and improve resolution of smaller proteins

Q5. The stacking gel in a discontinuous Tris–glycine SDS-PAGE system functions to:

• Focus proteins into a thin band via isotachophoresis before entering the resolving gel
• Immobilize proteins for in-gel digestion
• Raise ionic strength to reduce heating
• Chelate divalent cations to prevent nuclease activity

Correct Answer: Focus proteins into a thin band via isotachophoresis before entering the resolving gel

Q6. In SDS-PAGE, the dye front of bromophenol blue migrates approximately like a:

• 2–3 kDa peptide
• 25 kDa protein
• 100 kDa protein
• 1,000 kDa complex

Correct Answer: 2–3 kDa peptide

Q7. Regarding protein visualization sensitivity, which statement is most accurate?

• Silver staining is more sensitive (sub-ng) than Coomassie Brilliant Blue (tens of ng)
• Coomassie Brilliant Blue is more sensitive than silver staining
• Ponceau S is the most sensitive for in-gel detection
• Bromophenol blue provides ng-level sensitivity

Correct Answer: Silver staining is more sensitive (sub-ng) than Coomassie Brilliant Blue (tens of ng)

Q8. A safer, non-mutagenic alternative to ethidium bromide for staining DNA in agarose gels is:

• SYBR Safe
• Methylene blue
• Coomassie G-250
• DAPI

Correct Answer: SYBR Safe

Q9. In SDS-PAGE, a plot of log molecular weight versus relative mobility (Rf) typically shows:

• An approximately linear relationship over a defined size range
• A quadratic relationship across all sizes
• No correlation between mobility and size
• An inverse square relationship at all sizes

Correct Answer: An approximately linear relationship over a defined size range

Q10. A key advantage of capillary gel electrophoresis (CGE) over slab gels is:

• Higher efficiency and faster separations with minimal sample consumption
• Ability to separate intact tissues
• Requirement for no electric field
• Compatibility only with radioactive detection

Correct Answer: Higher efficiency and faster separations with minimal sample consumption

Q11. In isoelectric focusing using immobilized pH gradients, proteins migrate until:

• They reach the pH equal to their isoelectric point where net charge is zero
• They reach the anode regardless of charge
• Their hydrodynamic radius equals the pore size
• Their mass-to-charge ratio equals one

Correct Answer: They reach the pH equal to their isoelectric point where net charge is zero

Q12. Native PAGE is most appropriate when the goal is to:

• Preserve protein conformation and activity while separating by charge, size, and shape
• Determine accurate molecular weights of denatured proteins
• Resolve DNA fragments larger than 100 kb
• Detect phosphoproteins via Pro-Q Diamond staining after SDS-PAGE

Correct Answer: Preserve protein conformation and activity while separating by charge, size, and shape

Q13. To separate yeast chromosomal DNA (>1 Mb), the most suitable electrophoretic method is:

• Pulsed-field gel electrophoresis (PFGE)
• Standard agarose gel at constant field
• Capillary isoelectric focusing
• Blue native PAGE

Correct Answer: Pulsed-field gel electrophoresis (PFGE)

Q14. Increasing buffer ionic strength in a slab gel system typically:

• Increases Joule heating and can cause band distortion or smiling
• Decreases current and accelerates migration
• Prevents any pH changes and improves stacking
• Eliminates the need for cooling even at high voltage

Correct Answer: Increases Joule heating and can cause band distortion or smiling

Q15. In DNA loading dye for agarose gels, glycerol or sucrose is included primarily to:

• Increase sample density so it sinks into the well
• Provide reducing conditions to prevent oxidation
• Chelate metal ions that degrade DNA
• Intercalate into DNA and enhance fluorescence

Correct Answer: Increase sample density so it sinks into the well

Q16. Denaturing PAGE for RNA often includes 6–8 M urea to:

• Disrupt hydrogen bonding and maintain nucleic acids in a single-stranded state
• Promote duplex formation during electrophoresis
• Crosslink RNA to prevent degradation
• Increase fluorescence of ethidium bromide

Correct Answer: Disrupt hydrogen bonding and maintain nucleic acids in a single-stranded state

Q17. The “smiling” pattern of bands seen near the gel edges is most often caused by:

• Uneven heating and edge cooling leading to temperature gradients
• Excess reducing agent in the sample buffer
• Using freshly prepared buffers
• Over-polymerization of acrylamide due to too much TEMED

Correct Answer: Uneven heating and edge cooling leading to temperature gradients

Q18. In Western blotting, the step that immediately follows SDS-PAGE is:

• Electrotransfer of proteins onto a PVDF or nitrocellulose membrane
• Blocking the membrane with milk or BSA
• Incubation with primary antibody
• Chemiluminescent detection

Correct Answer: Electrotransfer of proteins onto a PVDF or nitrocellulose membrane

Q19. In agarose gels, for plasmid DNA of identical length, which form migrates fastest?

• Supercoiled (covalently closed circular) DNA
• Nicked open circular DNA
• Linear DNA
• All forms migrate identically

Correct Answer: Supercoiled (covalently closed circular) DNA

Q20. If a horizontal agarose gel is 12 cm between electrodes and you apply 120 V, the electric field strength is:

• 10 V/cm
• 1 V/cm
• 12 V/cm
• 120 V/cm

Correct Answer: 10 V/cm

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