SDS-PAGE provides a gel in which proteins have been separated out into bands corresponding to their sizes. Unless we are working with a pure solution of proteins, the resulting array of bands can be quite complex, and there is no way using routine staining methods to differentiate two proteins of a similar size.

Immuno-blotting techniques allow more specific identification of protein bands, using labeled antibodies targeted against our protein of interest.

The first stage of Western blotting after SDS-PAGE is to transfer the protein band to a suitable medium, typically a membrane made of nitrocellulose. This is best done using a “semi-dry electro-blotting” procedure, where the gel is placed over the membrane and then sandwiched between layers of moist filter paper. The sandwich is then placed between two flat electrodes with the membrane towards the positive electrode and clamped together. As the current flows, the proteins in the gel migrate across to the membrane.

Setting up a Western Blot

Once the protein bands have transferred to the nitrocellulose membrane, they must be visualized using labeled antibodies. The membranes are incubated in the presence of an unlabelled primary antibody which has been raised against the protein of interest in one species of animal (eg. a rabbit). This antibody bonds only to the protein it has been raised against.

Addition of primary antibodies

When the membrane is washed, the primary antibody will be washed away from all of the gel save the band of protein where it bound.

Washing away primary antibody

The membrane is then incubated in the presence of a secondary antibody raised against the antibodies of the first species (eg. if the first species was a rabbit, the secondary antibody might be an antibody against rabbit antibodies raised in a goat ie. goat-anti-rabbit). The secondary antibody will only bind to the primary antibodies, which are bound to the band representing the protein of interest.

Addition of secondary antibody

The secondary antibodies are labeled with a compound which creates a coloured product in the presence of another chemical. Once the membranes are washed and incubated with this colour-change compound, the protein will appear as a coloured band on the membrane.

Development of bands

The following online resources contain more information about western blotting (note that these are external sites which may not be accessible from some schools) :

  • Biosolutions animation on the principle behind the technique

  • University of Hawai’i video showing the technique performed