Protein purification and gel electrophoresis

What is protein purification?

The process of isolating an individual protein or complex from a mixture of cell components

Why is protein purification useful in research?

Allows protein structure and function to be studied

Why is protein purification useful in commercial environments?

May be useful as drugs

How can protein be denatured?

Using chaotropic agents or reducing agents

How and why can protein be detected using UV light?

Use a wavelength of ~280 nm. Aromatic rings of tryptophan and tyrosine absorb this light

How can size exclusion chromatography be used to purify proteins?

Beads containing small holes are present in the column. Smaller protein can pass through the holes so have a longer pathway and are present in later fractions than larger proteins

How can ion-exchange chromatography purify proteins?

Charged beads in the column can bind oppositely charged proteins due to the R-groups present

How can bound proteins be collected from ion-exchange chromatography?

Elute with increasing salt concentrations.
Lower concentrations remove weakly bound protein with smaller charges. Then increase concentration to remove other proteins

What type of ion-exchange chromatography isolates positive proteins?

Cation-exchange chromatography, uses negative beads

What type of ion-exchange chromatography isolates negative proteins?

Anion-exchange chromatography, uses positive beads

How can affinity chromatography be used to purify proteins?

Ligand with high affinity for the protein of interest is covalently attached to beads in a column

How can protein purity be measured?

Column chromatography methods, electrophoresis

How can column chromatography be used to determine protein purity?

Separation of proteins by molecular weight and determining the number present due to absorbance

What are the types of gel electrophoresis?

1D (SDS PAGE), 2D

How are proteins separated in 1D gel electrophoresis?

Due to molecular weight

How are proteins separated in 2D gel electrophoresis?

Due to isoelectric point and then molecular weight

What is the isoelectric point of a protein?

The pH at which the protein has no net charge.
Depends on the R-groups present

How can proteins be purified by SDS PAGE?

Protein denatured and treated with SDS. Voltage applied and proteins separate by denatured molecular weight

What is SDS, what does it do?

Sodium dodecyl sulphate. Causes denatured protein to form a rigid rod, produces a negative charge along the length of the protein with a constant charge:mass

Where do proteins move in SDS PAGE?

Down the vertical column, towards the anode

What can be added to denature proteins?

Urea, SDS, reducing agents

What is the action of urea during protein denaturation?

Disrupts non-covalent interactions in proteins, acts as a chaotrophe agent. Ensures monomers are present

What is a commonly used reducing agent, what is its function?

β-mercaptoethanol, reduces disulphide bridges.

What is the first step of western blotting?

SDS PAGE

How can western blotting be done?

SDS PAGE transferred to protein binding membrane. Blocking buffer added to the membrane. Primary antibody added, which will bind to a protein of interest. Secondary antibody added to detect the protein of interest.

What is the first stage of 2D gel electrophoresis?

Isoelectric focussing

How can proteins be separated by isoelectric focussing?

Proteins added to gel with an immobilised pH gradient. Proteins will migrate to the point where the pH=the proteins isoelectric point (pI)

If a protein is at a pH<pI what is its charge?

Positive

If a protein is at a pH>pI what is its charge?

Negative

What is the second stage of 2D gel electrophoresis?

SDS PAGE

What is proteomics?

Analysis of proteins expression

What is required for proteomics?

2D gel electrophoresis, mass spectrometry, database searching

How can changes in proteomics be determined from gels?

Using image analysis software