Description
Introduction: Heat shock proteins or HSPs are being synthesized under different kind of stress conditions and act as molecular chaperones for protein molecules. Because these proteins were first found in cells that were exposed to high temperatures, they are called "heat shock proteins" and have been named according to their molecular weights. Usually HSPs are cytoplasmic proteins and they function in various intra-cellular processes. Heat shock proteins play an important role in protein-protein interactions, including folding and assisting in establishing of proper protein conformation, and prevention of inappropriate protein aggregation. The HSP70 family is a set of highly conserved proteins that are induced by a variety of biological stresses, including heat stress, in every organism in which the proteins have been examined. The HSP70 family members include: HSP70, a 70 kDa protein which is strongly inducible in all organisms but which is also constitutively expressed in primate cells; HSP72, a 72 kDa protein which is induced exclusively under stress conditions; HSC70, or cognate protein, is a 72 kDa constitutively expressed protein which is involved in uncoating clathrin-coated vesicles; GRP78,or BiP, is a glucose-regulated 78 kDa protein localized to the endoplasmic reticulum; and mitochondrial HSP70 (mtHSP70, GRP75 or mortalin) a 75 kDa protein that is found within the mitochondria.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to HSP70. Standards or samples are then added to the appropriate microtiter plate wells with a biotin conjugated antibody preparation specific for HSP70 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain HSP70, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of HSP70 in the samples is then determined by comparing the O.D. of the samples to the standard curve