Biochemical Techniques and Methods Flashcards

SDS-PAGE separates proteins primarily based on:

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.

In a Western blot, after SDS-PAGE and transfer to membrane, what is the purpose of the primary antibody?

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.

The polymerase chain reaction (PCR) requires which enzyme?

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.

Which chromatographic technique separates molecules based on their specific binding interaction with an immobilized ligand?

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.

An ELISA (enzyme-linked immunosorbent assay) detects a target molecule using:

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.

Which technique is used to determine the three-dimensional structure of proteins at atomic resolution using X-rays?

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.