Chromatography

What is column chromatography used for?

Separating proteins based on how differently they migrate through a solid phase.

What is the mobile phase in column chromatography?

A buffered solution that migrates through the porous solid material.

What is the solid phase in column chromatography?

The porous material in the column that proteins migrate through.

What affects protein migration rate in column chromatography?

Protein properties.

What does ion-exchange chromatography separate by?

Sign and magnitude of net electric charge.

How does pH affect ion-exchange chromatography?

pH determines protein ionization state and therefore its net charge.

How does salt concentration affect ion-exchange chromatography?

It affects protein affinity for the ion-exchange resin.

What is a cation exchanger?

A resin with negatively charged groups that binds cations/positively charged proteins.

What is an anion exchanger?

A resin with positively charged groups that binds anions/negatively charged proteins.

Which protein moves faster on a cation exchanger?

A protein with a large net negative charge.

What is size-exclusion chromatography also called?

Gel filtration chromatography.

What does size-exclusion chromatography separate by?

Protein size, or molecular weight.

Which proteins emerge first in size-exclusion chromatography?

Large proteins emerge before small proteins.

Why do large proteins elute first in size-exclusion chromatography?

They are excluded from more of the pores and take a shorter path.

What does affinity chromatography separate by?

Binding affinity.

What is attached to the beads in affinity chromatography?

A ligand that specifically binds the protein of interest.

How is a protein commonly eluted from affinity chromatography?

With a high salt concentration or competing ligand.

What is electrophoresis used to do?

Visualize and characterize purified proteins.

What can protein electrophoresis estimate?

Protein number, degree of purity, isoelectric point, and approximate molecular weight.

What are protein electrophoresis gels made of?

Cross-linked polymer polyacrylamide.

What determines migration in general protein electrophoresis?

Charge-to-mass ratio.

How are proteins visualized after electrophoresis?

The gel is treated with a stain such as Coomassie blue.

What does Coomassie blue bind?

Proteins, but not the gel itself.

What is electrophoretic mobility (μ)?

The ratio of molecular velocity to electrical potential: μ = V/E.

How is electrophoretic mobility related to charge and friction?

μ = Z/f, where Z is net charge and f is the frictional coefficient.

What does the frictional coefficient reflect?

A protein's shape.

What factors affect protein migration in a gel?

Size and shape.

What does SDS stand for?

Sodium dodecyl sulfate.

What is SDS electrophoresis?

Denaturing electrophoresis used to estimate protein purity and molecular weight.

What does SDS do to proteins?

It binds and partially unfolds proteins.

Approximately how much SDS binds per amino acid residue?

Nearly one SDS molecule per residue.

What charge does SDS give proteins?

A large net negative charge.

Why does SDS make proteins separate mainly by molecular weight?

It gives proteins a similar charge-to-mass ratio.

Which proteins migrate faster during SDS electrophoresis?

Smaller proteins.

How can molecular weight be estimated from SDS electrophoresis?

Plot log(Mr) of markers versus relative migration; the relationship is linear.

What are marker proteins used for in SDS electrophoresis?

Known molecular-weight standards used to estimate an unknown protein's molecular weight.

What does UV absorbance reveal about aromatic amino acids?

Tryptophan, tyrosine, and phenylalanine absorb UV light.

Which aromatic amino acids contribute strongly near 280 nm?

Tryptophan and tyrosine.

What does circular dichroism (CD) spectroscopy measure?

Differences in absorption of left- vs right-handed circularly polarized light.

What is the primary CD chromophore in proteins?

The peptide bond (C–N).

What does CD spectroscopy assess in proteins?

Secondary structure.

What is the CD spectrum of an α-helix-rich native protein like?

Similar to the blue curve shown on the slide.

What happens to CD when a protein unfolds?

Defined secondary structure is lost and the spectrum resembles the black curve.

What electronic transitions are shown for the peptide bond in CD?

π→π* at 190–200 nm and n→π* at 210–220 nm.