Western Blotting

Proteins in extracts can be separated by size (fractionated) using polyacrylamide gel electrophoresis (PAGE). A reducing agent and an ionic detergent, sodium dodecyl sulphate (SDS), are included to keep the proteins denatured and negatively charged so that they are separated solely on the basis of their mass. The percentage acrylamide used is dependent on the size range of the proteins to be resolved - usually between 4% (large proteins) and 10% (smaller proteins) acrylamide gels are used.
SDS PAGE
Fig. 3-41, Lodish et al. 4th ed. 
By adjusting the % acrylamide, it is possible to resolve proteins within a tight range of sizes (e.g. protein which may be present in a phosphorylated and an unphosphorylated state - and differing in mass by only ~2 kDa*.) The fractionated proteins are electrophoretically transferred to a solid matrix such as nitrocellulose filter or nylon membrane and are immobilised in the exact same position as where they migrated in the gel.
Westerns  
Fig. 3-44, Lodish et al. 4th ed.

The protein of interest is detected with a specific antibody (just as for expression screening using a phage library, see Lecture , BB211 webnotes). The filter is incubated with the primary antibody, which will bind to the specific epitope it recognises.

After washing away the non-specifically bound antibody, the filters are usually incubated with a secondary antibody (produced in a different species of animal, raised against the IgGs from the species of animal in which the primary antibody was raised), which is fused to a reporter enzyme (alkaline phosphatase or horseradish peroxidase most commonly used) to allow detection of the antibody complex. The wash step is repeated and then the filter is incubated with the specific reagents for the reporter enzyme bound to the secondary antibody. This will result in a coloured reactant being deposited at the spot where the antibody complex is formed and provides a visual read-out of the exact location of the protein of interest.
The mass of the protein can be determined by comparing it to protein molecular weight standards which are run in a separate well of the gel alongside.
*Protein molecular mass is measured in Daltons (1 Da = 1 atomic mass unit - 1H)