Modern developments in Western blot techniques (lightning storms)
No commentsBy Gen Federico
Western blotting is a routine procedure in immunoassays. Proteins are first separated by gel electrophoresis, before being blotted onto a specialised membrane which binds with the protein. The membrane is blocked from further protein interaction, and is then incubated with antibodies targeted to the relevant antigens. These bind direct to the protein, hopefully with minimal interference from the substrate, producing a readable result.
Traditionally, protein detection has been a two-step, indirect process. The membrane-bound protein is incubated with a primary antibody, which is unlabelled and reacts with the antigen direct. The excess is then washed off and a secondary immunoglobulin applied, which binds to a certain site on the primary agent (i.e. an amino acid sequence). This is species-specific, and must be raised against the immunoblobulin of the species of the primary Ig. Secondary proteins are labelled (conjugated) with an enzyme or luminescent dye which, when activated, gives a readable signal.
Traditionally, a biotin-labelled secondary immunoglobulin is tagged with an enzyme such as horseradish peroxidise. This results in a reaction called DAB-staining which makes the proteins visible. They are then read in a spectrophotometer. Recently, fluorescent dyes such as DyLight have become a popular alternative. Here, the antibody is conjugated with a fluorescent probe which, when excited, fluoresces in the near-infra red range. Fluors are considered the most sensitive detection agents in blotting assays.
The two-step assay method remains popular because signals can be strongly amplified by using several secondary Igs, each of which bind to a different site on the primary structure. However, with improved detection methods and higher throughput techniques, interest has switched to one-step probing methods. This requires antibodies that recognise the target protein and are labelled for detectable protein tags.
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