HSP90ß (C-Terminal Domain) TR-FRET Kit

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Catalog #
50262
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Description

The HSP90β (C-Terminal Domain) TR-FRET Assay Kit is designed to measure binding activity of HSP90β (heat shock protein 90 kDa beta) to its target, PPID (peptidylprolyl isomerase D) for screening and profiling applications using TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer). It utilizes Terbium-labeled donor and a dye-labeled acceptor to complete the TR-FRET pairing. The HSP90β (C-Terminal Domain) TR-FRET Assay Kit comes in a convenient 96-well or 384-well format, with enough purified HSP90β (C-Terminal) amino acids 527-724), PPID, Tb-Labeled Donor and Dye-Labeled Acceptor and assay buffer for 96 or 384 reactions.

Figure 1: Illustration of the assay principle.
A sample containing terbium-labeled donor, dye-labeled acceptor, HSP90β (C-Terminal), PPID, and an inhibitor is incubated. The fluorescence intensity is then measured using a fluorescence reader. In the presence of binding of HSP90β (C-Terminal) to PPID, energy transfer occurs due to the proximity of the donor and acceptor. Disruption of the binding results in decrease of energy transfer. Fluorescence intensity at λ=665 nm corresponds directly to the binding of HSP90β (C-Terminal) to PPID. 

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Synonyms
HSP90b, Heat Shock Protein 90b, HSP90beta, HSP90β, HTRF
Product Info
Storage and Usage
Citations
Assay Kit Format
TR-FRET
Materials Required But Not Supplied
  • Adjustable micropipettor and sterile tips
  • Fluorescence plate reader capable of measurement TR-FRET. 
Format

96 Reactions:

Catalog # Name Amount Storage
50313 HSP90β (C-terminal), Biotin-Labeled, His-Tag, Avi-Tag* 5 µg -80°C
71095 PPID (CYP-40), GST-Tag 5 µg -80°C
  Tb-Labeled Donor 10 µl -20°C
  Dye-Labeled Acceptor 10 µl -20°C
50324 3x HSP90 Assay Buffer 2 4 ml -20°C
79685 Black 96-well microtiter plate 1 Room Temp

*The initial concentration of is lot-specific and will be indicated on the tube containing the protein

 

384 Reactions:

Catalog # Name Amount Storage 
50313 HSP90β (C-terminal), Biotin-Labeled, His-Tag, Avi-Tag* 10 µg -80°C
71095 PPID (CYP-40), GST-Tag 10 µg -80°C
  Tb-Labeled Donor 2 x 10 µl -20°C
  Dye-Labeled Acceptor 2 x 10 µl -20°C
50324 3x HSP90 Assay Buffer 2 4 ml -20°C
79685 White, nonbinding, low volume 384-well microtiter plate 1 Room Temp.

 

 

 

 

 

 

 

 

 

 

 

 

*The initial concentration of is lot-specific and will be indicated on the tube containing the protein.

UniProt #
P08238
Background

HSP90 (heat shock protein 90 kDa) is a member of the HSP family of proteins. HSP proteins are chaperone proteins, involved in aiding proteins to fold correctly and resist heat related stress, cell signaling by acting on hormone receptors and kinases, cytoskeleton organization, cell cycle and differentiation by acting on CDK4 (cyclin dependent kinase 4), Wee1 and CDK11p110, among others. HSP90 has 3 family members, which differ in their cellular localization. HSP90A is a cytosolic protein and has two inducible isoforms (HSP90α1 and α2) and one that is constitutively expressed (HSP90β). HSP90B (endoplasmin) is found in the endoplasmic reticulum, and TRAP (TNF receptor-associated protein 1) in the mitochondria. They are composed of four domains: N-terminal domain (NTD), linker, middle domain (MD) and a C-terminal domain (CTD). The NTD has a high affinity ATP binding site, while the CTD is mainly involved in target binding, dimerization and localization. HSP90β is regulated at the transcription level, by post-translational modifications such as phosphorylation, and by co-chaperones. HSP90 can lead to cancer progression, by participating in the stabilization of several oncogenes and proteins involved in angiogenesis, inflammation, tumor suppression and metastasis. HSP90 has thus become an attractive therapeutic target for cancer therapy. In addition to cancer, HSP90 can also play a role in Alzheimer’s disease, Parkinson disease and viral infection. It is clear from its multiple and crucial functions that the development of inhibitors targeting HSP90 can prove beneficial for the treatment of several debilitating and fatal diseases.

References

Allan R.K., et al., 2006 J. Biol. Chem. 281(11): 7161-71.
Hoter A., et al., 2018 Int J Mol Sci. 19(9):2560.