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BIOCHEMICAL TECHNIQUES USING ANTIBODY

The biochemical techniques using antibodies

In research, purified antibodies are used in many applications. They are most commonly used to identify and locate intracellular and extracellular proteins. Antibodies are used in flow cytometry to differentiate cell types by the proteins they express; different types of cell express the unusual combination of the cluster of differentiation molecules on their surface and produce different intracellular and secretable proteins. They also used in immunoprecipitation to separate proteins and anything bound to them (co-immunoprecipitation) from other molecules in a cell lysate.

In Western blot analysis to identify proteins separated by electrophoresis. By immunohistochemistry or immunofluorescence to examine protein expression in tissue sections or to locate proteins within cells with the assistance of a microscope. Protein can also be detected and quantified with antibodies, using ELISA (Enzyme Linked Immunosorbent Assay) and ELISPOT (Enzyme-Linked Immunosorbent Spot Techniques).

Method of analysis for DNA, RNA, and Protein:

  1. DNA: southern blot, PCR, Sequence analysis
  2. RNA: northern blot, RT-PCR
  3. Protein: western blot, ELISA, Immunoprecipitation, FACS, Histochemistry, etc.

 

Western blot

Western blot is often used to determine relative protein levels between samples. They also establish the molecular weight of the target, which may provide insight into its post-translational processing. Protein from tissue/cell lysates are separated by gel electrophoresis according to their molecular weight. Following separation samples are transferred to a membrane, where antibodies are applied to probe the protein of interest.

 

Immunoprecipitation (IP)

A method to study protein-protein interactions using an antibody. Principals:

  1. The antibody is allowed to bind to its antigen.
  2. The antibody is precipitated out of solution by protein A or protein G beads.
  3. The individual proteins in precipitates are separated by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis).
  4. Western blotting can be used to assess the proteins obtained.

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