Protein Purification

Why is protein purification important?

To determine the structure and mechanism of action of proteins.

What four properties are proteins separated based on during purification?

Charge, Size, Affinity, and Hydrophobicity.

What is the first step in protein purification?

Cell lysis to extract protein.

What is the goal of the protein purification workflow?

Maximum purity with minimum steps and resources.

What does ultracentrifugation analyze in proteins?

Shape, mass, and interactions.

What does the sedimentation velocity method measure?

Shape, mass, and interactions of proteins.

What does sedimentation equilibrium only measure?

Mass of the proteins.

What is the principle behind salting out?

Salt competes for water causing proteins to aggregate.

What separates proteins based on charge?

Ion Exchange Chromatography (IEX).

What is the Isoelectric Point (pI)?

The pH at which the net charge of a protein is zero.

What occurs at a pH greater than pI?

The protein is negatively charged.

What occurs at a pH less than pI?

The protein is positively charged.

How do cation exchange columns work?

They use negatively charged resin and operate at pH lower than pI.

What characterizes anion exchange chromatography?

It uses positively charged resin and operates at pH greater than pI.

What does affinity chromatography rely on?

Specific binding between the protein and an immobilized ligand.

Name two types of tags used in affinity chromatography.

Epitope tags and engineered tags.

What is the role of imidazole in IMAC?

To avoid non-specific binding during the process.

What does Size Exclusion Chromatography (SEC) separate by?

By size of the proteins.

What does high salt do in Hydrophobic Interaction Chromatography (HIC)?

Promotes binding of the hydrophobic proteins.

In 2D-PAGE, how are proteins separated?

First by charge (isoelectric focusing) and then by mass (SDS-PAGE).

What does SDS-PAGE separate proteins by?

Mass only, as it coats proteins with a uniform negative charge.

What are key terms in chromatography regarding phases?

Stationary phase (resin) and mobile phase (flowing liquid or buffer).

What is elution in chromatography?

Washing bound proteins off the column.

What is an eluate?

The collected sample after elution.

What is the significance of V₀ in chromatography?

Void volume, for excluded molecules.

What does ultracentrifugation help achieve in protein study?

It provides insights into mass and shape during analysis or preparation.

What do detergents do in membrane protein purification?

They form micelles around hydrophobic proteins to keep them soluble.

What is Lysozyme known for?

Cleaving peptidoglycan in bacterial cell walls.

What is a critical micelle concentration (CMC)?

The concentration of detergent needed to form micelles.

In the context of distribution in ion exchange chromatography, what pH corresponds to cation exchange?

pH less than pI, which allows negative resin to bind positively charged proteins.