Western blotting is a commonly used gel electrophoresis technique for the separation and detection of cellular proteins.
Samples containing a protein mixture (e.g. a cell lysate) are loaded into different wells of the polyacrylamide gel (one sample per well). An electric current is then applied that allows the (charged) proteins to travel along the gel to a distance that is inversely related to their molecular weight (MW, the bigger the protein the smaller distance it will travel along the gel). A MW size marker (ladder) is loaded either side of the samples on the gel to enable identification and confirmation of the size of the protein of interest.
The proteins within the gel are then transferred onto a blotting membrane, by applying a second electric current, at exactly the same position as they occupied on the gel relative to the MW size ladder.
The membranes are then treated with a blocking buffer (usually containing non-fat milk) followed by incubation with a primary antibody that will specifically bind the protein of interest (non-specific binding to other unrelated proteins is minimised with the blocking solution).
A secondary antibody linked to a molecular probe such as horse radish peroxidase is then applied to the membrane that will bind to the primary antibody (e.g. an anti-mouse or anti-rabbit antibody).
This binding can be detected via enhanced chemiluminescence by incubating the membranes with a mixture of hydrogen peroxide and luminol. The latter is cleaved in a coupled reaction to release blue light that can be detected, by exposure to an autoradiography film, in the form of dark bands corresponding to the site of antibody binding to the protein under investigation.
The bands are characterised by a) their vertical position, indicative of protein size (how far the protein traveled along the gel – this related to the MW size marker), and b) their relative intensity, indicative of the amount of protein present in each cell lysate.
In this way, we are able to identify patterns of protein expression (e.g. cleavage, increased or decreased amounts) associated with different cellular states (e.g. growth, apoptosis, drug treatment, etc.) by comparing samples loaded in adjacent lanes on the same gel (one sample in each lane).
