BIOLOGY 3594: Polyacrylamide Gel
Electrophoresis – Complete Set Up Guide 2026
Exam Questions with Correct Answers and
Detailed Rationales
,1. What is the primary purpose of polyacrylamide gel electrophoresis (PAGE)?
A. To separate nucleic acids by size
B. To separate proteins or nucleic acids based on size and charge
C. To amplify DNA sequences
D. To measure enzyme activity
Answer: B
Rationale: PAGE is a technique used to separate macromolecules (proteins, DNA,
RNA) primarily by their molecular weight and charge under an electric field.
2. In PAGE, the gel matrix is formed by the polymerization of which two
monomers?
A. Acrylamide and bis-acrylamide
B. Agarose and bis-acrylamide
C. Acrylamide and agarose
D. Bis-acrylamide and TEMED
Answer: A
Rationale: Acrylamide is the main monomer; bis-acrylamide
(N,N′-methylenebisacrylamide) acts as a cross-linker to create a three-dimensional
network.
3. The cross-linker in polyacrylamide gels is:
A. Acrylamide
B. Bis-acrylamide
C. TEMED
D. Ammonium persulfate (APS)
Answer: B
Rationale: Bis-acrylamide cross-links acrylamide chains, determining the pore size
of the gel.
4. Which two chemicals are used to initiate the polymerization of acrylamide?
A. Ammonium persulfate (APS) and TEMED
B. Acrylamide and bis-acrylamide
,C. SDS and β-mercaptoethanol
D. Coomassie Blue and acetic acid
Answer: A
Rationale: APS provides free radicals; TEMED (tetramethylethylenediamine)
catalyzes the formation of free radicals from APS.
5. The pore size of a polyacrylamide gel is primarily controlled by:
A. The voltage applied
B. The concentration of acrylamide and bis-acrylamide
C. The buffer pH
D. The running time
Answer: B
Rationale: Higher total acrylamide concentration (%T) gives smaller pores;
cross-linker ratio (%C) also affects pore size.
6. For separating large proteins (>200 kDa), which gel percentage is most
appropriate?
A. 15% acrylamide
B. 5% acrylamide
C. 10% acrylamide
D. 20% acrylamide
Answer: B
Rationale: Lower percentage gels have larger pores, allowing large proteins to
migrate more easily.
7. For separating small proteins (10–30 kDa), which gel percentage is typically
used?
A. 5%
B. 8%
C. 12-15%
D. 20%
, Answer: C
Rationale: Higher acrylamide concentration creates smaller pores, resolving
low-molecular-weight proteins.
8. In SDS-PAGE, the purpose of SDS (sodium dodecyl sulfate) is to:
A. Denature proteins and impart a uniform negative charge
B. Cross-link proteins
C. Reduce disulfide bonds
D. Stain the gel
Answer: A
Rationale: SDS binds to proteins at a constant ratio (1.4 g SDS per g protein),
masking intrinsic charge and making them negatively charged.
9. Which reducing agent is commonly added to sample buffer to break disulfide
bonds?
A. SDS
B. β-mercaptoethanol or dithiothreitol (DTT)
C. Glycerol
D. Bromophenol blue
Answer: B
Rationale: Reducing agents cleave disulfide bridges, fully denaturing proteins and
allowing separation by size alone.
10. The function of glycerol in the sample loading buffer is to:
A. Denature proteins
B. Increase sample density so it sinks into the well
C. Reduce disulfide bonds
D. Track the migration front
Answer: B
Rationale: Glycerol makes the sample heavier than the running buffer, preventing
it from floating out of the well.
Electrophoresis – Complete Set Up Guide 2026
Exam Questions with Correct Answers and
Detailed Rationales
,1. What is the primary purpose of polyacrylamide gel electrophoresis (PAGE)?
A. To separate nucleic acids by size
B. To separate proteins or nucleic acids based on size and charge
C. To amplify DNA sequences
D. To measure enzyme activity
Answer: B
Rationale: PAGE is a technique used to separate macromolecules (proteins, DNA,
RNA) primarily by their molecular weight and charge under an electric field.
2. In PAGE, the gel matrix is formed by the polymerization of which two
monomers?
A. Acrylamide and bis-acrylamide
B. Agarose and bis-acrylamide
C. Acrylamide and agarose
D. Bis-acrylamide and TEMED
Answer: A
Rationale: Acrylamide is the main monomer; bis-acrylamide
(N,N′-methylenebisacrylamide) acts as a cross-linker to create a three-dimensional
network.
3. The cross-linker in polyacrylamide gels is:
A. Acrylamide
B. Bis-acrylamide
C. TEMED
D. Ammonium persulfate (APS)
Answer: B
Rationale: Bis-acrylamide cross-links acrylamide chains, determining the pore size
of the gel.
4. Which two chemicals are used to initiate the polymerization of acrylamide?
A. Ammonium persulfate (APS) and TEMED
B. Acrylamide and bis-acrylamide
,C. SDS and β-mercaptoethanol
D. Coomassie Blue and acetic acid
Answer: A
Rationale: APS provides free radicals; TEMED (tetramethylethylenediamine)
catalyzes the formation of free radicals from APS.
5. The pore size of a polyacrylamide gel is primarily controlled by:
A. The voltage applied
B. The concentration of acrylamide and bis-acrylamide
C. The buffer pH
D. The running time
Answer: B
Rationale: Higher total acrylamide concentration (%T) gives smaller pores;
cross-linker ratio (%C) also affects pore size.
6. For separating large proteins (>200 kDa), which gel percentage is most
appropriate?
A. 15% acrylamide
B. 5% acrylamide
C. 10% acrylamide
D. 20% acrylamide
Answer: B
Rationale: Lower percentage gels have larger pores, allowing large proteins to
migrate more easily.
7. For separating small proteins (10–30 kDa), which gel percentage is typically
used?
A. 5%
B. 8%
C. 12-15%
D. 20%
, Answer: C
Rationale: Higher acrylamide concentration creates smaller pores, resolving
low-molecular-weight proteins.
8. In SDS-PAGE, the purpose of SDS (sodium dodecyl sulfate) is to:
A. Denature proteins and impart a uniform negative charge
B. Cross-link proteins
C. Reduce disulfide bonds
D. Stain the gel
Answer: A
Rationale: SDS binds to proteins at a constant ratio (1.4 g SDS per g protein),
masking intrinsic charge and making them negatively charged.
9. Which reducing agent is commonly added to sample buffer to break disulfide
bonds?
A. SDS
B. β-mercaptoethanol or dithiothreitol (DTT)
C. Glycerol
D. Bromophenol blue
Answer: B
Rationale: Reducing agents cleave disulfide bridges, fully denaturing proteins and
allowing separation by size alone.
10. The function of glycerol in the sample loading buffer is to:
A. Denature proteins
B. Increase sample density so it sinks into the well
C. Reduce disulfide bonds
D. Track the migration front
Answer: B
Rationale: Glycerol makes the sample heavier than the running buffer, preventing
it from floating out of the well.
