Proteins - Centrifugation & Chromatography

What is an exampled of a cation exchanger?

CM cellulose - negatively charged gel

What are the two phases of column chromatography?

1. The stationary phase - solid porou matrix supported by a column 2. The mobile phase - percolates over stationary phase fed by reservoir, protein sample applied in this phase

Sedimentation via centrifugation is dependent on...?

- Size of particle - Shape of particle - Density of particle - Density of solution being centrifuged

What is ion exchange chromatography?

It is a process that allows the separation of polar molecules base on their affinity to the ion exchanger.

What is centrifugation?

It is a process that involes the use of the centrifugal force for the sedimentation of heterogenous mixtures with a centrifuge.

What is hydrophobic chromatography?

It is the same principle as 'salting out'. The high salt content dehydrates protein. Gel matrix composed of hydrophobic groups - binds to hydrophobic domains in protein.

What is density gradient centrifugation?

It is used to separate proteins with different sedimentation coefficients. It typically separates proteins differing in sedimentation coefficients by a factor of two or more. Further purification often required.

Explain size exclusion chromatography

Mobile phase is run through a gel composed of porous beads of an insoluble polymer. Gels are available in a wide range of pore sizes. Large proteins are excluded from pores and small proteins are retained in pores.

How can proteins be fractionated by size and shape?

Single filter - fixed pore size. Molecular sieve - limited use.

How is the bound protein eluted?

The bound protein must then be eluted by either: - Increasing ionic strength of buffer - competitively disrupts the ionic interactions bewteen protein and resin - Alteration of pH - cause interacting groups of protein to lose their charge

What is the ideal pH of the mobile phase and why?

The mobile phase pH should be between the pKa of the solid phase and the pKa of the protein of interest.

What are the two types of ion-exchangers?

Anion exchangers bind anions to a positive column Cation exchangers bind cations to a negative column

What are the 4 principles types of column chromatography used to purify proteins?

1. Size exclusion 2. Ion exchange 3. Affinity 4. Hydrophobic

What is an example of an anion exchanger?

DEAE cellulose - positively charged gel

True or false: small proteins are eluted first and large proteins are eluted last

False Large proteins are excluded from pores so are eluted first. Small proteins are eluted last

Explain the process of affinity chromatography

I.e a protein that has high affinity for glucose. Column composed of beads linked to glucose, only glucose binding protein is retained on the gel. Eluted by increasing the concentration of glucose.

What is differential centrifugation?

Provides several fractions (different components of mixture) of decreasing density. It is the starting point for subsequent purification techniques. Generally smaller components require longer and higher speeds.

What are Svedberg units?

This a sedimentation coefficient. It is a measure reflective of size and shape.

What is affinity chromatography?

This separates proteins on the basis of the binding affinity for specific chemical groups. Any ligand that binds to protein specifically can be used i.e. antibody, metal, cofactor. Provides very high resolution of purification.

True or False: Proteins are eluted at the base of the column and collected. It exploits differences in properties of proteins.

True

True or False: consider a mix of proteins applied to a cation exchange column, negatively charged proteins pass through more rapidly and elute earlier while positively charged proteins remained bound.

True

True or False: For large macromolecules to be fractionated, supernatant needs to be subject to very-high-speed centrifugation

True.

Density gradient centrifugation is divided into velocity sedimentation and equilibrium sedimentation. Explain the difference.

Velocity sedimentation - shallow gradient used which allows sedimentation of different components with different S values Equilibrium sedimentation - steep gradient used to separate based on density. Allows separation with high sensitivity.