Immunodetection


Immunodetection is an umbrella term for the techniques used to detect specific proteins in a mixture, using complementary antibodies which are labelled for detection. Immunodetection can be performed either in vitro in a homogenate sample or in vivo in a cell or tissue.

The antibodies used in immunodetection can be monoclonal, a single antibody species derived from a clonal cell line, or polyclonal, where there is a population of different antibodies that recognise different parts of the protein. Antibodies are obtained by inoculating a mouse with an antigen, extracting immune cells and fusing them with tumour cells to produce clonal antibody-producing cell lines called hybridomas.

The antibodies are labelled for detection either radioactively (125I), fluorescently using FITC (green) or rhodamine (red), or by conjugation to a fluorophore or an enzyme that catalyses a bioluminescent reaction.

Techniques include:

For the in vitro detection of a specific protein:
  • Immuno dot blots
  • Western blotting (or immunoblotting)
  • Immunoprecipitation (also allows detection of post-translational modifications)

For the in vivo detection of a specific protein:
  • Immunocytochemistry
  • Immunohistochemistry

For the in vivo detection of protein-DNA interactions:
  • Chromatin immunoprecipitation (chIP)

For monitoring of in vivo protein synthesis:
  • Pulse-chase

For the in vitro detection of protein-protein interactions
  • GST pull-down experiments