Western blot analysis MCQs With Answer

Introduction: Western blot analysis is an essential technique in immunotechnology for detecting specific proteins within complex biological samples. This quiz set is tailored for M.Pharm students to reinforce advanced understanding of principles, experimental design, troubleshooting, and quantitative aspects of Western blotting. Questions cover sample preparation, SDS-PAGE, membrane selection, transfer methods, blocking strategies, antibody usage, detection systems (chemiluminescence and fluorescence), normalization, and common artifacts. By practicing these MCQs, you will sharpen critical thinking for assay optimization, interpretation of band patterns, and validation of results—skills necessary for research, quality control, and therapeutic protein characterization.

Q1. Which of the following statements best explains why sodium dodecyl sulfate (SDS) is used during sample preparation for standard Western blotting?

• SDS cross-links proteins to preserve native quaternary structure
• SDS provides a uniform negative charge proportional to protein size and denatures secondary and tertiary structures
• SDS prevents proteins from entering the gel by increasing their molecular weight
• SDS enhances antigen–antibody binding during membrane probing

Correct Answer: SDS provides a uniform negative charge proportional to protein size and denatures secondary and tertiary structures

Q2. Which membrane is generally preferred when reprobing (stripping and re‑probing) multiple times for low-abundance targets and why?

• Nitrocellulose, because it has higher protein binding capacity and better mechanical strength
• PVDF, because it has higher protein binding capacity and superior chemical/mechanical durability after repeated stripping
• Cellulose acetate, because it prevents nonspecific antibody binding
• Polyethersulfone, because it increases chemiluminescent signal sensitivity

Correct Answer: PVDF, because it has higher protein binding capacity and superior chemical/mechanical durability after repeated stripping

Q3. Which transfer method is most appropriate for very large proteins (>250 kDa) to ensure efficient transfer from gel to membrane?

• Semi-dry transfer at high current for short time
• Wet (tank) transfer at low voltage for extended time with cold buffer circulation
• Electroblotting using dry transfer membranes like iBlot for 7–10 minutes
• Capillary transfer using filter paper without electric field

Correct Answer: Wet (tank) transfer at low voltage for extended time with cold buffer circulation

Q4. During Western blot blocking, which blocker is least recommended when probing phospho-proteins with anti-phospho-specific antibodies and why?

• 5% non-fat dry milk, because it contains casein (a phosphoprotein) that can increase background and compete with antibody binding
• 3% BSA, because it is expensive compared to milk
• Commercial proprietary blocking buffer, because it reduces sensitivity for all antibodies
• 1% gelatin, because it enhances signal for phospho-specific antibodies

Correct Answer: 5% non-fat dry milk, because it contains casein (a phosphoprotein) that can increase background and compete with antibody binding

Q5. Which of the following describes the primary advantage of using HRP-conjugated secondary antibodies with enhanced chemiluminescent (ECL) substrates?

• Fluorescent detection avoids substrate depletion seen with HRP
• HRP + ECL provides high sensitivity with short exposure times and broad dynamic range suitable for low-abundance proteins
• HRP allows direct visualization without need for antibodies
• ECL substrates permanently stain the membrane for downstream mass spectrometry

Correct Answer: HRP + ECL provides high sensitivity with short exposure times and broad dynamic range suitable for low-abundance proteins

Q6. When performing quantitative Western blotting, which approach best ensures accurate normalization across lanes?

• Using a single housekeeping protein (e.g., β-actin) without validation of its expression stability
• Staining the membrane for total protein (e.g., Ponceau S or total protein fluorescent stain) and using that for normalization
• Normalizing only to molecular weight marker intensities
• Adjusting exposure to saturate the target band for easier detection

Correct Answer: Staining the membrane for total protein (e.g., Ponceau S or total protein fluorescent stain) and using that for normalization

Q7. Which gel percentage is most appropriate to separate proteins in the range of 20–40 kDa with good resolution?

• 4% polyacrylamide gel
• 6% polyacrylamide gel
• 10–12% polyacrylamide gel
• 20% polyacrylamide gel

Correct Answer: 10–12% polyacrylamide gel

Q8. What is the primary purpose of including a reducing agent (e.g., β-mercaptoethanol or DTT) during sample preparation for SDS-PAGE?

• To maintain disulfide bonds and preserve native protein conformation
• To reduce oxidized SDS molecules and improve gel polymerization
• To break disulfide bonds, ensuring subunits separate and migrate according to polypeptide size
• To prevent proteins from binding to the membrane during transfer

Correct Answer: To break disulfide bonds, ensuring subunits separate and migrate according to polypeptide size

Q9. If you observe high background across an entire Western blot membrane after detection, which of the following is the most likely cause?

• Insufficient blocking or use of an inappropriate blocking agent
• Overly diluted primary antibody
• Running the gel at too low a voltage
• Using too high a percentage gel for low-molecular-weight proteins

Correct Answer: Insufficient blocking or use of an inappropriate blocking agent

Q10. When selecting a primary antibody for detecting a post-translational modification, which factor is most critical?

• That the antibody recognizes the linear amino acid sequence irrespective of modification state
• That the antibody was raised against a peptide containing the specific modified residue and validated for that modification
• That the antibody is monoclonal regardless of epitope specificity
• That the antibody is conjugated to HRP to avoid need for a secondary antibody

Correct Answer: That the antibody was raised against a peptide containing the specific modified residue and validated for that modification

Q11. Why is methanol commonly used to pre-wet PVDF membranes before transfer?

• Methanol activates PVDF by removing SDS from proteins after transfer
• Methanol hydrates and wets the hydrophobic PVDF surface, improving protein binding and handling
• Methanol prevents gel polymerization during electrophoresis
• Methanol blocks nonspecific antibody binding sites permanently

Correct Answer: Methanol hydrates and wets the hydrophobic PVDF surface, improving protein binding and handling

Q12. Which control would best demonstrate antibody specificity in a Western blot experiment?

• Loading unequal amounts of lysate across lanes
• Using a pre-immune serum as primary antibody negative control and a knockout cell lysate lacking the target protein as an additional specificity control
• Exposing the blot for excessively long times to reveal faint bands
• Using multiple secondary antibodies in the same incubation

Correct Answer: Using a pre-immune serum as primary antibody negative control and a knockout cell lysate lacking the target protein as an additional specificity control

Q13. In chemiluminescent detection, what factor most commonly limits the quantitative linear range of signal detection?

• The concentration of SDS in the sample buffer
• The depletion or saturation of chemiluminescent substrate and detector dynamic range, causing nonlinear signals at high protein loads
• The choice of molecular weight marker
• The use of PVDF vs nitrocellulose

Correct Answer: The depletion or saturation of chemiluminescent substrate and detector dynamic range, causing nonlinear signals at high protein loads

Q14. What is the recommended approach if a target band appears at a higher-than-expected molecular weight?

• Assume the result is correct and publish without further validation
• Consider post-translational modifications, alternative splice forms, glycosylation, or incomplete reduction; treat sample with deglycosylation enzymes or use reducing/denaturing conditions and verify with controls
• Always switch to a higher percentage gel to shift the band downward
• Ignore molecular weight markers during interpretation

Correct Answer: Consider post-translational modifications, alternative splice forms, glycosylation, or incomplete reduction; treat sample with deglycosylation enzymes or use reducing/denaturing conditions and verify with controls

Q15. During semi-dry transfer, which parameter should be carefully optimized to prevent poor transfer or overheating?

• Blocking buffer composition
• Transfer current, time, and buffer ionic strength to balance efficient transfer without heating or gel drying
• Primary antibody dilution
• Percentage of stacking gel only

Correct Answer: Transfer current, time, and buffer ionic strength to balance efficient transfer without heating or gel drying

Q16. Which technique improves multiplex detection when using fluorescently labeled secondary antibodies?

• Using secondary antibodies from the same host species for all primaries
• Selecting spectrally distinct fluorophores and using primary antibodies raised in different host species or directly conjugated primaries to avoid cross-reactivity
• Probing with HRP-conjugated secondaries after fluorescence imaging
• Stripping and reprobing between each fluorescent detection step only

Correct Answer: Selecting spectrally distinct fluorophores and using primary antibodies raised in different host species or directly conjugated primaries to avoid cross-reactivity

Q17. What is the main benefit of using a protease inhibitor cocktail in cell lysate preparation for Western blotting?

• To denature proteins for better migration on the gel
• To prevent degradation of proteins during lysis and sample handling, preserving integrity of target proteins
• To improve antibody binding on the membrane
• To increase gel polymerization speed

Correct Answer: To prevent degradation of proteins during lysis and sample handling, preserving integrity of target proteins

Q18. Which of the following troubleshooting steps is most appropriate if you observe a single strong band at the expected size plus multiple faint lower-molecular-weight bands (proteolytic fragments)?

• Increase incubation time with primary antibody
• Add or increase protease inhibitors, work at lower temperatures, and minimize freeze–thaw cycles to reduce proteolysis
• Switch to nitrocellulose membrane to eliminate fragments
• Remove reducing agent from sample buffer to prevent fragmentation

Correct Answer: Add or increase protease inhibitors, work at lower temperatures, and minimize freeze–thaw cycles to reduce proteolysis

Q19. For direct comparison of protein expression between samples on different gels, which practice yields the most reliable quantitative data?

• Comparing band intensity visually without normalization
• Including a common internal standard or reference sample on every gel and normalizing signals to that standard and to total protein per lane
• Using different exposure times for each gel to optimize contrast
• Running samples in different buffer systems to improve separation

Correct Answer: Including a common internal standard or reference sample on every gel and normalizing signals to that standard and to total protein per lane

Q20. Which statement most accurately describes the advantage of fluorescent Western blot detection over chemiluminescent detection for quantitative applications?

• Fluorescence always has higher sensitivity than chemiluminescence for ultralow-abundance targets
• Fluorescent detection allows multiplexing with multiple non-overlapping fluorophores and provides a wider linear dynamic range with stable signals for more accurate quantitation
• Fluorescent methods do not require secondary antibodies
• Fluorescent detection eliminates the need for blocking

Correct Answer: Fluorescent detection allows multiplexing with multiple non-overlapping fluorophores and provides a wider linear dynamic range with stable signals for more accurate quantitation

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