Chromatography

What is the purpose of protein purification?

To isolate and enrich a specific protein from a complex mixture.

Why might scientists purify a protein?

For structural analysis, biochemical assays, or therapeutic use.

What is needed to identify a target protein throughout purification?

A highly sensitive and specific biochemical assay.

What does a biochemical assay measure during protein purification?

The activity of the target protein in each sample or fraction.

What reaction does firefly luciferase catalyze?

An ATP-dependent reaction that converts D-luciferin into oxyluciferin and emits light.

Why is luciferase useful as a biochemical assay?

The emitted light provides a sensitive way to track luciferase activity during purification.

What is the first step in preparing proteins from cells?

Disrupting the cells to release their contents.

What methods can be used to disrupt cells?

Sonication, shearing, or treatment with mild detergents.

Why must cell disruption conditions be gentle?

To preserve the target protein’s function.

How can researchers determine whether protein function was preserved during cell disruption?

By using a biochemical assay.

What does preparative centrifugation separate cellular components based on?

Size and density.

What two basic fractions are produced by centrifugation?

The pellet and the supernatant.

What happens when centrifugal force and centrifugation time are progressively increased?

Different subcellular components are separated into fractions.

What cellular fractions can be obtained through differential centrifugation?

Nuclei, mitochondria, membranes, and cytosol.

What is specific activity?

The target protein’s activity units divided by the total amount of protein.

What is the formula for specific activity?

Specific activity = activity units ÷ total protein.

What does specific activity indicate?

The purity of the target protein.

What should happen to specific activity as a protein is purified?

It should increase because contaminating proteins are being removed.

How is fold-purification calculated?

Specific activity after purification ÷ initial specific activity.

If 90% of the original activity and 25% of the original protein remain, what is the fold-purification?

0.90 ÷ 0.25 = 3.6-fold purification.

What is the basic principle of column chromatography?

Proteins are separated based on their different physical or chemical interactions with a solid gel matrix.

Which proteins elute first from a chromatography column?

Proteins that interact poorly or weakly with the matrix.

What is a fraction in column chromatography?

A portion of liquid collected as proteins elute from the column.

How is the target protein located among the collected fractions?

Each fraction is tested using a biochemical assay.

What happens to fractions containing the highest yield of the target protein?

They are combined, or pooled.

What property separates proteins in gel-filtration chromatography?

Protein size.

What is the gel-filtration matrix made from?

Porous carbohydrate beads made of agarose.

Which proteins elute first in gel-filtration chromatography?

The largest proteins.

Why do large proteins elute first in gel-filtration chromatography?

They cannot enter as many pores in the beads, so they travel through the column more directly.

Which proteins elute last in gel-filtration chromatography?

The smallest proteins.

Why do small proteins elute later in gel-filtration chromatography?

They enter the pores of the beads, giving them a longer route through the column.

What analogy describes gel-filtration chromatography?

It is like a race through an obstacle course in which the largest proteins finish first.

What property separates proteins in ion-exchange chromatography?

Overall electrical charge.

What does a cation-exchange column bind?

Positively charged proteins.

What charge does a cation-exchange column carry?

A negative charge.

What does an anion-exchange column bind?

Negatively charged proteins.

What charge does an anion-exchange column carry?

A positive charge.

What happens to proteins that do not bind the ion-exchange matrix?

They wash through the column.

How can proteins bound to an ion-exchange column be eluted?

By adding an excess of like-charged ions or changing the pH.

Why does changing pH affect ion-exchange chromatography?

A protein’s overall charge is pH-dependent.

What is DEAE cellulose?

A positively charged matrix that binds negatively charged proteins.

Is DEAE cellulose a cation exchanger or an anion exchanger?

An anion exchanger because it binds negatively charged proteins.

What is carboxymethylcellulose, or CMC?

A negatively charged matrix that binds positively charged proteins.

Is CMC a cation exchanger or an anion exchanger?

A cation exchanger because it binds positively charged proteins.

Why can the terms “cation exchanger” and “anion exchanger” be confusing?

The name describes the charge of the protein or ion that the matrix binds, not the charge of the matrix itself.

What happens after proteins are eluted from an ion-exchange column?

The fractions are assayed for the target protein, and the appropriate fractions are pooled.

What property separates proteins in affinity chromatography?

A specific binding interaction between the target protein and a ligand.

How is an affinity chromatography column constructed?

A specific ligand is attached to beads in the column.

What happens to the target protein in affinity chromatography?

It binds specifically to the ligand attached to the beads.

What happens to nonspecific proteins in affinity chromatography?

They wash through the column.

How can a target protein be eluted from an affinity column?

By adding a competing ligand, adding salt, or changing the pH.

Why is affinity chromatography highly selective?

It uses a ligand that specifically binds the target protein.

When are His-tags particularly useful?

When a convenient natural ligand–protein interaction is unavailable.

What is a His-tag?

An engineered sequence containing six or more histidine residues attached to a recombinant protein.

What type of column binds a His-tagged protein?

A Ni²⁺-NTA affinity column.

What part of a His-tagged protein interacts with the Ni²⁺ column?

The histidine residues.

How can a His-tagged protein be eluted from a Ni²⁺ affinity column?

By using imidazole or a low-pH solution.

Why does imidazole elute a His-tagged protein?

It competes with the histidine residues for interaction with the Ni²⁺ column.

What may happen to the His-tag after the protein is purified?

It may be removed using a protease.

What are the three common types of column chromatography?

Gel filtration, ion exchange, and affinity chromatography.

What does gel-filtration chromatography separate proteins by?

Size.

What does ion-exchange chromatography separate proteins by?

Overall charge.

What does affinity chromatography separate proteins by?

A specific ligand-binding interaction.

Why are multiple purification techniques often combined?

Different methods remove different contaminants, producing greater overall enrichment of the target protein.

How much enrichment can a multistep purification scheme achieve?

Several thousand-fold purification of the target protein.

What measurements should be made after each purification step?

Target-protein activity and total protein, which are used to calculate specific activity.

What indicates that a purification step was successful?

An increase in the target protein’s specific act