Chapter 3 Part 2 - Chromatography

What characteristic does salting out take advantage of?

solubility of protein

What characteristic does ion exchange take advantage of?

charge

What characteristic does gel filtration take advantage of?

size

What characteristic does affinity chromatography take advantage of?

binding specificity

When more salt is added in ammonium salt precipitation, what is happening to the POI?

It’s hydration later is being disrupted

What is the purpose of column chromatography?

To measure the interaction of analyte between the stationary and mobile phase

What charge must the stationary phase be in cation exchange?

negative

What charge must the stationary phase be in anion exchange?

Positive

Type of cation and anion exchange chromatography

Cation - CM cellulose

Anion - DEAE

How do you elute a protein in cation/anion chromatography?

Gradually increase salt concentration in mobile phase

How do you elute in size-exclusion/gel-filtration?

Add buffer

What is the relationship to PI and overall charge?

If pH = PI, no net charge

If pH < PI, favor protonation, (+) charge

If pH > PI, favor deprotonation, (-) charge

At what point is a proteins PI?

At the equivalence point

What elutes first and last in an immobilized metal chelate affinity (IMAC)?

First: compounds without His-tag

Last: compounds with His-tag

How to elute in IMAC?

Add imidazole to mobile phase

What metals are used in metal chelate affinity?

Fe, Co, Ni, Cu

What elutes first and last in affinity chromatography?

First: compounds with low affinity for binding partner attached to stationary phase

Last: compounds with high affinity for binding partner attached to stationary phase

How to elute in affinity chromatography

Add compound with high affinity for stationary phase to mobile phase. The compound with the higher affinity for the stationary phase will bind to it and allow the mobile phase to elute out.

How does SDS-PAGE allow us to characterize a protein?

SDS is a detergent that makes proteins negatively charged. This denatures the protein, making it lose its structure and migrate towards the positive pole

How does PAGE allow us to characterize a protein?

It applies a current to separate proteins based on size where the smallest proteins migrate the furthest (kDa)

What does PAGE measure?

The purity of a protein

What dye is used in PAGE to visualize the sample?

Coomassie

How does isoelectric focusing work?

• Proteins are loaded on the negatively charge high pH side.

• Proteins migrate to low pH positively charged side until the pH = PI (no net charge)

• At high pH, ALL proteins are negative so they all migrate

What is 2D-PAGE?

using (1) isoelectric focusing to separate by PI and (2) SDS page to separate by MW

What is a protein assay?

A measurement that can detect the total activity of protein, amount of substrate converted to product and enzyme rate of reaction

It can also detect total amount of protein by:

• Absorbance at 280 nm

• Bradford assay

• Western blot

• and ELISA

What is an enzyme

A biocatalyst that speeds up reaction by lowering activation energy

What happens to protein activity as you purify it?

Less activity

which amino acids absorb light and at what absorbance

Tryptophan and tyrosine at 280 nm

How can a Bradford assay be used to quanitify your protein?

The bradford dye binds to all proteins nonspecifically but binds to K and R. When it is bound to protein, it turns blue. When it does not it turns brown/red.

At what absorbance value does bradford dye show up?

595 nm

How can a western blot be used to quantify your POI?

A primary antibody that is specific to the protein binds to POI. A secondary antibody is added that binds to the primary antibody and allows for visualization of protein amount.

Darker bands = more protein

What is an ELISA assay?

An enzyme-linked immunosorbent assay that detects the exact amount of a specific protein desired using an antibody. No purification necessary.

What is the stationary phase?

The resin, what the protein interacts with

What is the mobile phase?

Proteins in buffer

What are fractions in column chromatography?

Small volumes of eluent collected in 1 mL increments.

How does IMAC chromatography work?

A stationary phase with immobilized metal ions to capture proteins with a natural affinity for those metals or with a histidine-rich tag.

• During the binding phase, the sample is passed through a column, and target proteins attach to the metal ions while other molecules flow through.

• To elute the proteins, a solution is introduced that competes with the protein for the metal ions, such as a high concentration of imidazole, or the pH is altered.

Which two protein purification techniques can be affected by pH?

1. Ammonium sulfate precipitation

2. Ion exchange chromatography

What is the purpose of adding salt in ion exchange chromatography?

The salt dissociates into ions and displaces the proteins on the column