Hsp70 and Hsp90-alpha Detection Kits

These kits allow direct measurements using a fixed end-point assay based on immunofluorescence detection in cells grown on standard high-density microplates. The DNA binding dye, DAPI, enables nuclear size and morphology determination and cell cycle phase identification by DNA content. The primary antibodies are specific for their targets and have minimal cross-reactivity.

Heat shock proteins (HSP) are essential for protein folding, protein synthesis, cellular stress defense and many other functions. Cellular stress increases the HSP levels in cells by transcriptional regulation through HSF-1, STAT1, ATF3 and c Jun, which is critical to maintain cellular homeostasis. Induction of HSP is closely correlated with substance cytotoxicity and lipophilicity given to the cell. Hsp70 is involved in guiding protein translocation across organelle membranes, disassembling oligomeric protein structures, facilitating proteolytic degradation of unstable proteins and controlling the biological activity of folded regulatory proteins. Nuclear localization of Hsp70 is associated with cell stress and cell growth.

Hsp90 is one of the most abundant proteins in eukaryotic cells. Hsp90 has chaperone activity with many other co-chaperones and is important for folding proteins of various signaling pathways. The two isoforms of Hsp90, alpha and beta, have the same function. Because the altered chaperone function of HSP is associated with the development of many human diseases including cancer, neurodegenerative disorders, and cardiovascular disease, making chaperones targets for drug development.

The Hsp70 and Hsp90α Detection Kits have been optimized with the Thermo Scientific ArrayScan® HCS Reader using the Compartmental Analysis BioApplication Software Module. Thus, automated plate-handling, focusing, cell image acquisition and processing, and data analysis and management are combined in one high-content screening (HCS) system to assay for test compounds. In addition to HCS instruments, cells labeled by the kit reagents can be viewed and analyzed by fluorescence and confocal microscopes.

Figure 2. Hsp70, Hsp90a and nuclei staining in A549 cells (20X objective). Cells were non-treated or treated with CuSO4 (1 mM) for 24 hours, stained as described in the protocol and imaged using the ArrayScan® V^TI HCS Reader.

 
Figure 3. Chemical induction of Hsp70 and Hsp90αin A549 cells. Cells were treated with different concentrations of chemicals (top panel: CuSO₄ bottom panel: menadione and ZnCl₂). Each data point represents 16 wells from three separate plates. Hsp70 and Hsp90α were measured using the output parameter of nuclear fluorescence or the difference between cytoplasm and nucleus average fluorescence intensity (cyt-nuc diff) with Cellomics Compartmental Analysis Bioapplication. Values were normalized with the maximum control value. EC₅₀ values are listed.