GTP Protein Detection and Techniques and Application 10 M

What does protein spectroscopy measure and how?

Protein concentration by measuring UV light absorption.

What is the Beer-Lambert Law formula and what does each variable mean?

A = εcl; A = absorbance, ε = extinction coefficient, c = concentration, l = path length.

What is the purpose of SDS in SDS-PAGE?

denatures proteins and gives them a uniform negative charge.

How does SDS-PAGE separate proteins?

By molecular weight as they migrate through a gel in an electric field.

What is isoelectric focusing?

A technique that separates proteins based on their isoelectric point

What is 2D electrophoresis and why is it useful?

Combines isoelectric focusing and SDS-PAGE to separate proteins by both charge and size.

Which amino acids are chromophoric and absorb UV light?

Tryptophan, tyrosine, and phenylalanine.

What is the extinction coefficient?

A value that describes how strongly a molecule absorbs light at a specific wavelength.

What is Western blotting used for?

To detect specific proteins using antibodies after gel separation.

Why is a blocking step used in Western blotting?

To prevent nonspecific antibody binding to the membrane.

What is the role of the primary antibody in Western blotting?

It binds directly to the target protein.

What is the role of the secondary antibody?

It binds to the primary antibody and provides a detectable signal (e.g., via enzyme or fluorescence).

What’s the difference between monoclonal and polyclonal antibodies?

Monoclonal bind a single epitope; polyclonal bind multiple epitopes on the same antigen.

What are limitations of protein analysis techniques like spectroscopy and Western blotting?

Spectroscopy lacks specificity and can be affected by contaminants; Western blotting is specific but expensive and depends on antibody quality.

Which amino acids are chromophoric and absorb UV light?

Tryptophan, tyrosine, and phenylalanine.