SDS-PAGE, ELISA and Western blotting principles MCQs With Answer

SDS-PAGE, ELISA and Western blotting are foundational techniques in cellular and molecular pharmacology, essential for protein analysis, quantification, and validation. This quiz-focused blog provides M.Pharm students with targeted multiple-choice questions that probe underlying principles, experimental design, troubleshooting, and interpretation of results. Emphasis is placed on mechanistic understanding — how SDS confers uniform charge, how gel composition affects resolution, distinctions among ELISA formats and their analytical implications, and critical steps in Western blot transfer, detection and quantitation. Use these MCQs to test conceptual depth, prepare for exams, and reinforce best practices for laboratory experiments involving protein separation, immunodetection and data reliability.

Q1. What is the primary basis for separation of proteins in SDS-PAGE?

• Intrinsic net charge of native proteins
• Three-dimensional conformation of proteins
• Relative molecular weight (size) of denatured proteins
• Isoelectric point (pI) of proteins

Correct Answer: Relative molecular weight (size) of denatured proteins

Q2. What is the main role of SDS (sodium dodecyl sulfate) in PAGE?

• To crosslink polyacrylamide chains
• To coat proteins with a uniform negative charge proportional to length
• To enzymatically digest proteins into peptides
• To act as a fluorescent stain for proteins

Correct Answer: To coat proteins with a uniform negative charge proportional to length

Q3. Why is a reducing agent like beta-mercaptoethanol or DTT included in sample buffer for SDS-PAGE?

• To increase gel pore size
• To preserve disulfide bonds and native folding
• To break disulfide bonds and fully denature multi‑subunit proteins
• To stain proteins for visualization

Correct Answer: To break disulfide bonds and fully denature multi‑subunit proteins

Q4. What is the function of the stacking gel in discontinuous SDS-PAGE systems?

• To separate proteins strictly by isoelectric point
• To concentrate and sharpen protein bands before entering resolving gel
• To polymerize acrylamide faster than the resolving gel
• To provide fluorescent background for imaging

Correct Answer: To concentrate and sharpen protein bands before entering resolving gel

Q5. How does increasing acrylamide percentage in the resolving gel affect protein separation?

• Higher percentage improves resolution of large proteins (>200 kDa)
• Higher percentage improves resolution of small proteins (<30 kDa)
• Acrylamide percentage has no effect on resolution
• Higher percentage decreases gel mechanical stability

Correct Answer: Higher percentage improves resolution of small proteins (<30 kDa)

Q6. What is the primary purpose of including a molecular weight marker (protein ladder) on an SDS-PAGE gel?

• To act as an internal loading control for quantitation
• To estimate the molecular masses of separated proteins
• To bind antibodies during Western blotting
• To accelerate polymerization of acrylamide

Correct Answer: To estimate the molecular masses of separated proteins

Q7. Which staining method is more sensitive for detecting low-abundance proteins on a gel?

• Coomassie Brilliant Blue staining
• Silver staining
• Ponceau S staining
• Ethidium bromide staining

Correct Answer: Silver staining

Q8. How does isoelectric focusing (IEF) differ fundamentally from SDS-PAGE?

• IEF separates proteins by molecular weight; SDS-PAGE separates by isoelectric point
• IEF separates proteins by isoelectric point (pI); SDS-PAGE separates by molecular weight
• Both techniques separate proteins by hydrophobicity
• IEF requires SDS to impart uniform charge while SDS-PAGE does not

Correct Answer: IEF separates proteins by isoelectric point (pI); SDS-PAGE separates by molecular weight

Q9. Which description best fits a sandwich ELISA?

• An antigen is directly labeled and detected without antibodies
• A primary antibody is labeled and binds directly to antigen coated on plate
• An antigen is captured by a plate-bound antibody and detected by a second antibody
• Unlabeled antigen competes with labeled antigen for antibody binding sites

Correct Answer: An antigen is captured by a plate-bound antibody and detected by a second antibody

Q10. What is the advantage of using an indirect ELISA format over a direct ELISA?

• It eliminates the need for secondary antibodies
• It gives greater signal amplification because a labeled secondary antibody recognizes the primary antibody
• It requires fewer incubation steps than direct ELISA
• It measures enzyme activity directly without antibodies

Correct Answer: It gives greater signal amplification because a labeled secondary antibody recognizes the primary antibody

Q11. Which chromogenic substrate is commonly used with horseradish peroxidase (HRP) in ELISA to produce a colorimetric signal?

• BCIP/NBT
• TMB (3,3′,5,5′-Tetramethylbenzidine)
• Alkaline phosphatase luminescent substrate
• Coelenterazine

Correct Answer: TMB (3,3′,5,5′-Tetramethylbenzidine)

Q12. What is the main purpose of blocking the membrane or ELISA plate with BSA, nonfat dry milk, or casein?

• To increase antigenicity of target proteins
• To prevent nonspecific binding of antibodies and reduce background
• To denature proteins for better transfer
• To act as an enzyme substrate during detection

Correct Answer: To prevent nonspecific binding of antibodies and reduce background

Q13. Which transfer method is generally most effective for efficient transfer of very high molecular weight proteins from gel to membrane?

• Tank (wet) electroblot transfer
• Semi-dry transfer for 5 minutes
• Passive capillary transfer without electric field
• Direct imaging of gel without transfer

Correct Answer: Tank (wet) electroblot transfer

Q14. Compared to nitrocellulose, what is a key advantage of PVDF membranes for Western blotting?

• PVDF has lower protein binding capacity than nitrocellulose
• PVDF is more fragile and cannot be handled multiple times
• PVDF has higher protein binding capacity and is better for reprobing
• PVDF automatically emits chemiluminescence without substrate

Correct Answer: PVDF has higher protein binding capacity and is better for reprobing

Q15. What chemical reaction underlies ECL (enhanced chemiluminescence) detection in Western blotting using HRP?

• HRP catalyzes oxidation of luminol producing light
• Alkaline phosphatase cleaves a chromogenic phosphate producing color
• Radioactive decay from labeled antibodies emits photons
• Fluorescent dye absorbs and emits light directly

Correct Answer: HRP catalyzes oxidation of luminol producing light

Q16. Which of the following is commonly used as a loading control for normalization in Western blot analysis?

• Bacterial RNase
• Beta-actin
• Horseradish peroxidase
• ELISA substrate

Correct Answer: Beta-actin

Q17. Which method is considered a relatively gentle stripping procedure to remove antibodies from a Western blot for reprobing?

• Heating in 2% SDS with beta-mercaptoethanol at 95°C for 30 minutes
• Incubation in low-pH glycine buffer (e.g., pH ~2.2) with mild detergent
• Treating membrane with proteases to remove bound antibodies
• Exposing membrane to concentrated NaOH

Correct Answer: Incubation in low-pH glycine buffer (e.g., pH ~2.2) with mild detergent

Q18. In a competitive ELISA, how does the signal intensity relate to the concentration of antigen in the sample?

• Signal intensity is directly proportional to antigen concentration
• Signal intensity is inversely proportional to antigen concentration
• Signal intensity is independent of antigen concentration
• Signal intensity increases only after antigen saturation

Correct Answer: Signal intensity is inversely proportional to antigen concentration

Q19. When designing a sandwich ELISA, what is an important requirement for the pair of capture and detection antibodies?

• Both antibodies must bind to the identical epitope
• They should recognize two distinct, non‑overlapping epitopes on the antigen
• Both must be polyclonal from the same species for specificity
• The detection antibody must be unlabeled to avoid interference

Correct Answer: They should recognize two distinct, non‑overlapping epitopes on the antigen

Q20. Which practice is essential for reliable quantitative comparison of protein levels by Western blot?

• Using saturated chemiluminescent exposure for maximum signal
• Ensuring target and loading control signals fall within the linear dynamic range and normalizing accordingly
• Comparing only band intensities without any normalization
• Collecting single replicate data to avoid variability

Correct Answer: Ensuring target and loading control signals fall within the linear dynamic range and normalizing accordingly

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