Western blot

Southern and Northern blots detect specific DNA and RNA. But genes ultimately do their work as proteins, and you often want to know whether a particular protein is present in a sample and how much there is. The Western blot answers that — it is the protein member of the blotting family, and it swaps the DNA probe for an antibody.

First, proteins from a cell extract are separated by size on a gel (SDS-PAGE coats them with detergent so they migrate purely by size). Then they are transferred onto a membrane, preserving the pattern. Now comes the protein-specific twist: instead of a complementary nucleic-acid probe, you use an antibody — an immune protein that binds one specific target protein with great selectivity (molecular recognition by shape, not by base-pairing). A primary antibody sticks to your protein of interest; a secondary antibody carrying an enzyme or fluorescent tag then binds the primary, amplifying the signal. Wherever the antibody bound, a band appears. The band's position confirms the protein's size, and its intensity reflects how much protein is present.

The Western blot is one of the most-used techniques in all of biology and medicine. It confirms that a gene you cloned is actually being translated into protein, checks whether a protein is more or less abundant under a treatment, and detects post-translational changes like phosphorylation (with antibodies specific to the modified form). It is also a familiar diagnostic confirmation test — historically used to confirm HIV antibodies. Its limit is specificity: a result is only as trustworthy as the antibody, which can bind off-target proteins and give misleading bands.