Description

Nori Equine HSP90 ELISA Kit Summary

Alternative names for HSP90: Heat shock protein 90

Alternative name for equine: Horse

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background:

Heat shock protein 90 (Hsp90) is a chaperone protein that assists other proteins to fold properly, stabilizes proteins against heat stress, and aids in protein degradation. It also stabilizes a number of proteins required for tumor growth, which is why Hsp90 inhibitors are investigated as anti-cancer drugs. Heat shock proteins, as a class, are among the most highly expressed cellular proteins across all species.^[1] As their name implies, heat shock proteins protect cells when stressed by elevated temperatures. They account for 1–2% of total protein in unstressed cells. However, when cells are heated, the fraction of heat shock proteins increases to 4–6% of cellular proteins.^[2] This protein was first isolated by extracting proteins from cells stressed by heating, dehydrating or by other means, all of which caused the cell’s proteins to begin to denature.^[3] However it was later discovered that Hsp90 also has essential functions in unstressed cells. The Hsp90 protein contains three functional domains, the ATP-binding, protein-binding, and dimerizing domain, each of which playing a crucial role in the function of the protein. The glucocorticoid receptor (GR) is the most thoroughly studied example of a steroid receptor whose function is crucially dependent on interactions with Hsp90.^[51][52]  Hsp90 stabilizes various growth factor receptors^[59] and some signaling molecules including PI3K and AKT proteins. Hence inhibition of Hsp90 may induce apoptosis through inhibition of the PI3K/AKT signaling pathway and growth factor signaling generally.^[4] One important role of Hsp90 in cancer is the stabilization of mutant proteins such as v-Src, the fusion oncogene Bcr/Abl, and mutant forms of p53 that appear during cell transformation. It appears that Hsp90 can act as a “protector” of less stable proteins produced by DNA mutations.^[5] Hsp90 is also required for induction of vascular endothelial growth factor and nitric oxide synthase.^[25]

References

1. Csermely P, et al. (1998). Ther. 79 (2): 129–68.
2. Crevel G, et al. (2001). Cell. Sci. 114 (Pt 11): 2015–25.
3. Prodromou C, et al. (2000). EMBO J. 19 (16): 4383–92.
4. Grad I, Picard D (2007). Cell. Endocrinol. 275 (1–2): 2–12.
5. Calderwood SK, et (2006). Trends Biochem. Sci. 31 (3): 164–72.
6. Fontana J, et al. (2002). Res. 90 (8): 866–73.

Product Citation