A western blot (alternately, immunoblot) is a method to detect a specific
protein in a given sample of tissue homogenate or extract. It uses
gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) (Figure 1) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically
nitrocellulose or
PVDF), where they are probed (detected) using
antibodies specific to the target protein. There are now many reagent companies that specialise in providing antibodies (both
monoclonal and
polyclonal antibodies) against many thousands of different proteins. This has dramatically reduced the time to carry out a blot. Previously large animals (e.g. sheep, goat - lots of serum) had to be immunised with the target protein twice (secondary immune response generates high affinity antibodies). Then either serum could be purified and used (polyclonal antibodies) or B cells could be isolated from the animal and fused
in vitro with mouse myeloma cells to generate
hybridomas that then provided single-specificity antibodies (monoclonal antibodies). Commercial antibodies are expensive, though the unbound antibody can be reused between experiments. This method is used in the fields of
molecular biology,
biochemistry,
immunogenetics and other molecular biology disciplines.
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