Hsp27 and Phospho-Hsp27 Detection Kits

These kits allow direct measurements of Hsp27 modulation and Hsp27 phosphorylation 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. The anti-phospho-Hsp27 antibody detects at Ser78.

Heat shock proteins (HSP) are essential for protein folding, protein synthesis, cellular stress defense and many other functions. Cellular stress increases HSP levels in cells by transcriptional regulation through HSF 1, STAT1, ATF3 and c-Jun. This cellular response is critical to maintain cellular homeostasis. The altered expression level of small HSP is associated with many human diseases including cancer, cataracts, neurodegenerative disorders and cardiovascular disease. Hsp27 (HSBP1, Hsp20, Hsp25 or Hsp28) forms monomers, dimers and homotypic oligomers and binds to other proteins, possibly acting as ATP-independent chaperones. HSP27 interacts with different proteins implicated in the apoptotic process and helps maintain structural integrity and membrane stability.

The Hsp27 and phospho-Hsp27 Detection Kits were 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. In addition to HCS instruments, cells stained by the kit reagents can be viewed and analyzed by fluorescence and confocal microscopes.

     
Figure 2. Staining of A549 cells (20X objective). The cells were treated media only (non-treated) or with CuSO₄ (1 mM), stained according to the kit protocol and imaged using the ArrayScan® V™ HCS Reader. DNA content (nuclei) was detected with DAPI (blue); Hsp27 primary antibody was detected with DyLight™ 488 (green) secondary antibody; Phospho-Hsp27 was detected with DyLight 549 (orange) secondary antibody.
 
 
 

Figure 3. Drugs induce Hsp27 and phospho-Hsp27 in the cytoplasm and nucleus. A549 cells were treated with different concentrations of drugs (CuSO4, menadione, ZnCl2, anisomycin). Each data point represents 16 wells from three plates. Hsp27 and phospho-Hsp27 were measured using the output parameter of cytoplasmic (cyto) or nuclear fluorescence (nuc) 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.