W - BIOL200 - 2.6 PROTEIN CHARACTERIZATION

What do chromogenic-enzyme assays detect?

Enzyme presence and activity via color change.

What do antibody assays detect?

Protein amount using labeled antibodies.

What is GFP tagging?

Tagging proteins with Green Fluorescent Protein to visualize expression.

What does Western blotting combine?

Gel electrophoresis + protein detection using antibodies.

 What are the 3 steps of Western blotting?

Run SDS-PAGE, bind primary antibody, detect with fluorescent secondary antibody.

What is immunoprecipitation (IP)?

 Pulling specific proteins from mixtures using antibody-antigen specificity.

How does IP work?

Add antibody → bind protein → add agent to precipitate complex.

What does SDS-PAGE do?

Separates proteins based on size using negatively charged SDS.

Steps of SDS-PAGE?

Denature → coat with SDS → load gel → apply current → separate by size.

Which proteins move faster in SDS-PAGE?

Smaller proteins.

What does 2D electrophoresis separate by?

 First charge (IEF), then size (SDS-PAGE).

What is isoelectric focusing (IEF)?

Separates proteins until they reach their pI (net charge = 0).

What does mass spec detect?

Mass-to-charge ratio (m/z) of proteins.

 Key components of mass spec?

 Ion source, mass analyzer, strike detector, computerized data system.

Is mass spec sensitive?

Yes, detects down to 1×10⁻¹⁵ mol and distinguishes similar proteins.

 What does mass spec give?

Relative amount of protein, not absolute.

How does peptide mass spec work?

Peptides → ionize → fragment → analyze fragments → get sequence.

Which technique detects light vs. heavy nitrogen (WIZE-14 vs. WIZE-15)?

Mass spec.