Immunoblotting techniques – ELISA, Western blotting and Southern blotting MCQs With Answer

Immunoblotting techniques – ELISA, Western blotting and Southern blotting MCQs With Answer

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Introduction

Immunoblotting techniques such as ELISA, Western blotting and Southern blotting are essential laboratory tools for B.Pharm students to detect and analyze biomolecules. ELISA (enzyme-linked immunosorbent assay) quantifies antigens or antibodies using plate-based immunoassays; Western blotting separates proteins by SDS-PAGE and detects specific proteins on membranes (nitrocellulose or PVDF) using primary and secondary antibodies; Southern blotting identifies specific DNA fragments after restriction digestion and gel electrophoresis by hybridization with labeled probes. Understanding sample preparation, blocking, probes, detection methods (colorimetric, chemiluminescent, fluorescent), controls and troubleshooting improves experimental design and data interpretation. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the fundamental principle of an ELISA?

  • An enzyme-conjugated antibody produces a measurable signal upon substrate addition
  • Separation of proteins by size on a gel
  • Hybridization of a labeled DNA probe to a target fragment
  • Use of restriction enzymes to cut DNA

Correct Answer: An enzyme-conjugated antibody produces a measurable signal upon substrate addition

Q2. Which membrane is commonly used for protein transfer in Western blotting?

  • Nitrocellulose
  • Agarose
  • Polyacrylamide gel
  • Cellulose acetate

Correct Answer: Nitrocellulose

Q3. Southern blotting is mainly used to detect which biomolecule?

  • DNA fragments
  • Proteins
  • Polysaccharides
  • Lipids

Correct Answer: DNA fragments

Q4. Which blocking agent is commonly used to prevent non-specific binding in Western blots?

  • Non-fat dry milk
  • SDS
  • EDTA
  • RNase A

Correct Answer: Non-fat dry milk

Q5. In a sandwich ELISA, which component captures the antigen?

  • Capture antibody coated on the plate
  • Substrate added after incubation
  • Secondary antibody conjugated to enzyme
  • Labeled DNA probe

Correct Answer: Capture antibody coated on the plate

Q6. Which enzyme-substrate pair is commonly used for colorimetric ELISA detection?

  • HRP and TMB
  • Restrictase and agarose
  • Kinase and ATP
  • Lyase and pH indicator

Correct Answer: HRP and TMB

Q7. What is the role of SDS in SDS-PAGE prior to Western blotting?

  • Denatures proteins and confers a uniform negative charge
  • Cross-links proteins to membranes
  • Lyses DNA for Southern blotting
  • Acts as a substrate for HRP

Correct Answer: Denatures proteins and confers a uniform negative charge

Q8. Which detection method produces light that can be captured on film or a CCD camera in Western blotting?

  • Chemiluminescence using HRP and luminol
  • Colorimetric TMB on ELISA plates
  • Ethidium bromide staining of DNA
  • Silver staining of lipids

Correct Answer: Chemiluminescence using HRP and luminol

Q9. Who is credited with developing the Southern blot technique?

  • Ed Southern
  • James Watson
  • Paul Ehrlich
  • Gregor Mendel

Correct Answer: Ed Southern

Q10. Which wash buffer additive reduces non-specific binding in ELISA and Western blotting?

  • Tween-20
  • Ethidium bromide
  • Sodium dodecyl sulfate at high concentration
  • Proteinase K

Correct Answer: Tween-20

Q11. What is the main purpose of a secondary antibody in Western blotting?

  • Bind to the primary antibody and carry a detection enzyme or label
  • Directly bind antigen epitopes
  • Denature proteins before electrophoresis
  • Digest unbound probes

Correct Answer: Bind to the primary antibody and carry a detection enzyme or label

Q12. Which ELISA format is best for measuring antibody concentration in serum?

  • Indirect ELISA
  • Southern ELISA
  • Competitive PCR
  • Gel filtration ELISA

Correct Answer: Indirect ELISA

Q13. For Southern blotting, what is the purpose of denaturation of DNA in the gel before transfer?

  • Convert double-stranded DNA to single strands for probe hybridization
  • Fix proteins to the membrane
  • Stain DNA with Coomassie blue
  • Activate restriction enzymes

Correct Answer: Convert double-stranded DNA to single strands for probe hybridization

Q14. Which membrane is preferred when reprobing Western blots multiple times?

  • PVDF because it has high protein-binding capacity and is durable
  • Agarose because it is porous and reversible
  • Nylon because it dissolves easily
  • Cellulose acetate because it is low-binding

Correct Answer: PVDF because it has high protein-binding capacity and is durable

Q15. What is a common loading control used to confirm equal protein loading in Western blots?

  • β-actin
  • Random DNA ladder
  • HRP enzyme
  • Alkaline phosphatase substrate

Correct Answer: β-actin

Q16. In Southern blotting, which factor increases hybridization stringency?

  • Higher temperature and lower salt concentration
  • Lower temperature and higher salt concentration
  • Increasing agarose concentration in gel
  • Adding milk as a blocking agent

Correct Answer: Higher temperature and lower salt concentration

Q17. Which substrate yields a blue/purple precipitate commonly used for alkaline phosphatase detection?

  • BCIP/NBT
  • TMB
  • Luminol
  • Ethidium bromide

Correct Answer: BCIP/NBT

Q18. What is one advantage of sandwich ELISA over direct ELISA?

  • Higher specificity and sensitivity due to two antibodies recognizing the antigen
  • Requires only one antibody, making it faster
  • Detects DNA fragments instead of proteins
  • Does not require washing steps

Correct Answer: Higher specificity and sensitivity due to two antibodies recognizing the antigen

Q19. During Western blot transfer, what is the purpose of methanol in transfer buffer?

  • Stabilize proteins and improve binding to nitrocellulose
  • Denature DNA to single strands
  • Act as a substrate for HRP
  • Digest antibody Fc regions

Correct Answer: Stabilize proteins and improve binding to nitrocellulose

Q20. Which labeling method is traditionally used for probes in Southern blot detection?

  • Radioactive isotopes (e.g., 32P) or non-radioactive labels like DIG
  • HRP conjugation to antibodies
  • Fluorescent proteins expressed in bacteria
  • Gold nanoparticle staining

Correct Answer: Radioactive isotopes (e.g., 32P) or non-radioactive labels like DIG

Q21. What does a competitive ELISA measure differently compared to other ELISA types?

  • Analyte concentration inversely related to signal due to competition
  • Only qualitative presence/absence of antigen
  • Direct detection of DNA sequences
  • Protein molecular weight distribution

Correct Answer: Analyte concentration inversely related to signal due to competition

Q22. Which step is unique to Southern blotting but not to Western blotting?

  • Hybridization of a labeled DNA probe to immobilized nucleic acids
  • Blocking with non-fat dry milk to prevent non-specific antibody binding
  • Incubation with primary and secondary antibodies
  • Protein denaturation with SDS

Correct Answer: Hybridization of a labeled DNA probe to immobilized nucleic acids

Q23. What is the primary reason to include a molecular weight marker in SDS-PAGE and Western blotting?

  • Estimate the molecular weight of target proteins
  • Act as a blocking agent
  • Increase transfer efficiency
  • Serve as enzyme substrate

Correct Answer: Estimate the molecular weight of target proteins

Q24. If you obtain high background in a Western blot, which action is most appropriate?

  • Increase blocking time or change blocking agent
  • Reduce agarose gel percentage
  • Use a stronger restriction enzyme
  • Skip the wash steps to save time

Correct Answer: Increase blocking time or change blocking agent

Q25. Which instrument is used to read absorbance in a plate-based ELISA?

  • Microplate reader (spectrophotometer)
  • PCR thermocycler
  • Gel documentation system for ethidium bromide
  • Mass spectrometer

Correct Answer: Microplate reader (spectrophotometer)

Q26. In Western blotting, what does a strong band at unexpected size commonly indicate?

  • Protein degradation or post-translational modification or cross-reactivity
  • Correct antibody specificity and perfect sample prep
  • Successful DNA hybridization
  • High ELISA optical density

Correct Answer: Protein degradation or post-translational modification or cross-reactivity

Q27. Which of the following improves sensitivity in chemiluminescent Western detection?

  • Use of enhanced chemiluminescent (ECL) substrate and optimized antibody concentrations
  • Eliminating primary antibody incubation
  • Using unblocked membrane to increase signal
  • Shortening gel run time drastically

Correct Answer: Use of enhanced chemiluminescent (ECL) substrate and optimized antibody concentrations

Q28. What is the effect of too much antibody in an ELISA?

  • High background or non-specific signal
  • Loss of DNA bands in Southern blot
  • Complete inhibition of enzyme activity
  • Improved gel polymerization

Correct Answer: High background or non-specific signal

Q29. Which step follows restriction digestion in a Southern blot protocol?

  • Gel electrophoresis to separate DNA fragments
  • Incubation with primary antibody
  • Coating ELISA plate with capture antibody
  • Blocking membrane with BSA for protein detection

Correct Answer: Gel electrophoresis to separate DNA fragments

Q30. For quantification in ELISA, which component is essential to generate a standard curve?

  • Known concentrations of the target analyte (standards)
  • Random DNA ladder
  • Only a single positive control sample
  • Excess secondary antibody without substrate

Correct Answer: Known concentrations of the target analyte (standards)

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