Biochemistry Biochemical Techniques and Methods 1 — Questions and Answers
Question 1: SDS-PAGE separates proteins primarily based on:
- Net charge at physiological pH
- Molecular weight (size) (Correct answer)
- Isoelectric point
- Hydrophobicity
Correct answer: Molecular weight (size)
SDS denatures proteins and coats them with uniform negative charge proportional to mass, so migration in the polyacrylamide gel reflects molecular weight only.
Question 2: In a Western blot, after SDS-PAGE and transfer to membrane, what is the purpose of the primary antibody?
- To block non-specific binding sites
- To specifically recognize the target protein (Correct answer)
- To generate a detectable signal directly
- To transfer protein from gel to membrane
Correct answer: To specifically recognize the target protein
The primary antibody binds specifically to the target protein (antigen) on the membrane, providing specificity; a secondary antibody conjugated to an enzyme or fluorophore then generates the detectable signal.
Question 3: The polymerase chain reaction (PCR) requires which enzyme?
- RNA polymerase
- Reverse transcriptase
- Thermostable DNA polymerase (Taq polymerase) (Correct answer)
- DNA ligase
Correct answer: Thermostable DNA polymerase (Taq polymerase)
PCR uses a thermostable DNA polymerase (typically Taq from Thermus aquaticus) that retains activity through the high-temperature denaturation cycles required to separate DNA strands.
Question 4: Which chromatographic technique separates molecules based on their specific binding interaction with an immobilized ligand?
- Size-exclusion chromatography
- Ion-exchange chromatography
- Affinity chromatography (Correct answer)
- Reverse-phase HPLC
Correct answer: Affinity chromatography
Affinity chromatography exploits specific, reversible binding between a target molecule and an immobilized ligand (e.g., His-tag/Ni-NTA, antibody/antigen) to achieve high-purity protein isolation.
Question 5: An ELISA (enzyme-linked immunosorbent assay) detects a target molecule using:
- Radioactively labeled antibodies (RIA method)
- Enzyme-conjugated antibodies producing a colorimetric signal (Correct answer)
- Fluorescent dyes that stain all proteins nonspecifically
- Electric current to detect protein migration
Correct answer: Enzyme-conjugated antibodies producing a colorimetric signal
ELISA uses antibodies conjugated to enzymes (e.g., HRP or alkaline phosphatase) that convert a substrate into a colored product, allowing quantitative detection of antigens.
Question 6: Which technique is used to determine the three-dimensional structure of proteins at atomic resolution using X-rays?
- Nuclear magnetic resonance (NMR) spectroscopy
- Cryo-electron microscopy (cryo-EM)
- X-ray crystallography (Correct answer)
- Circular dichroism (CD) spectroscopy
Correct answer: X-ray crystallography
X-ray crystallography diffracts X-rays through protein crystals and uses the resulting diffraction pattern to calculate electron density maps, from which atomic coordinates are determined.
Biochemistry Biochemical Techniques and Methods 1
SDS-PAGE separates proteins primarily based on: