Question

Describe the similarities and differences between HPLC, Fluorescent, ELIZA, and western blotting. Specifically, in type of information that each technique provides us about proteins.

Answer 1

The similarities among HPLC, fluorescent ELISA and Western blotting is that all these methods are used for detection and purification of proteins. HPLC is used to elute and purify proteins using a high pressure, ELISA and Western blot used antibodies against the desired protein to detect and quantify the protein.

The differences among HPLC, fluorescent ELISA and Western blotting are in the methodology and the protein detection limit. The detection limit of HPLC varies from ng/ µl to pg/µl while for ELISA and Western blot it is pg/µl. In HPLC, the proteins are eluted according to their hydrophobicity and can then be lyophilized. ELISA uses antibody linked to an enzyme to produce a detectable signal in the form of a colour change. Western blot combines gel electrophoresis and probing with specific antibodies to detect the desired proteins.  Western blotting is a more sensitive technique than ELISA.