Western Blotting

What is western blotting?

A protein detection technique.

What does western blotting utilise?

Specific antibodies to detect a protein of interest.

What are the 3 main steps?

SDS-PAGE - separates proteins in a gel according to size.

Protein transfer onto a high-affinity protein-binding matrix.

Sequential incubations with primary and secondary antibodies to detect target protein.

What happens during SDS-PAGE?

Protein mixtures are denatured in SDS solution by boiling.

SDS gives each protein an overall negative charge.

A polyacrylamide gel is made that resolves the protein according to size (proteins migrate towards positive electrode).

Larger proteins get retarded in the gel more than smaller ones.

What happens in protein transfer?

Once proteins have been resolved in the gel, they are subject to electro-transfer onto a membrane, such as nitrocellulose

What happens in western blotting?

Step-wise procedure involving specific antibodies to detect your protein of interest.

What are the steps within western blotting?

Membrane blocking, primary antibody incubation, secondary antibody incubation, detection of bound Ab complex.

What is membrane blocking?

Nitrocellulose membrane has a very high affinity for protein.

Therefore, milk proteins non-specifically bind and block aberrant antibody binding in subsequent steps.

What is primary antibody incubation?

Antibody incubated with membrane. If all goes well, the primary antibody should bind to its target epitope on protein of interest.

What is secondary antibody incubation?

Secondary antibodies are formulated to bind specific species of primary antibodies. Oestrogen receptor Ab is a Mouse, therefore we require a Rabbit anti-Mouse secondary antibody to bind the primary oestrogen receptor antibody. The secondary antibody is conjugated to horse-radish peroxidase enzyme.

What is detection of bound Ab complex?

The horse-radish peroxidase conjugate emits light upon substrate addition.

What are some control options?

Proteins such as a-Tubulin and B-actin as they are expressed at equal levels in cells and thus represent a useful way of monitoring loading between samples.

What is another control for western analysis?

As an addition to house-keeping protein analysis, protein concentrations of each sample can be determined using a spectrophotometer (Bradford assay) prior to loading on a gel. Once each sample concentration is known, equal quantities of protein can be loaded onto a gel, in theory negating the need to perform western analysis using a-tubulin.