Immunophilin FKBP52 Detection Kit

This kit allows 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 anti-FKBP52 antibody is specific with minimal cross-reactivity.

Figure 2. Staining of FKBP52 and nuclei in 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^TI HCS Reader.

This kit detects inducible FKBP52 (also known as FKBP4, FKBP59, Hsp56, Hsp59) protein in the cell. FKBP52 is a large immunophilin that binds to the immunosuppressive drug FK506 and has peptidyl-prolyl cis-trans isomerase (PPIase) activity, which is inhibited by the binding of FK506. Immunophilins are enriched in the central and peripheral neurons and reports indicate that FKBP52 has neurotrophic activity. FKBP52 contains three tetratricopeptide repeat (TPR) domains that mediate protein-protein interactions with Hsp90 and a calmodulin-binding site in the C-terminal region. In addition to neuroprotection, FKBP52 is involved in the maturation and translocation of steroid hormone receptors, transcription regulation, copper transport and cardiotrophic effect.

This kit was 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 labeled by the kit reagents can be viewed and analyzed by fluorescence and confocal microscopes.

 

Figure 3. Chemicals induce FKBP52 in A549 cells. Different chemicals (Top panel: CuSO₄ Bottom panel: menadione and ZnCl₂ ) were added to the cells at the indicated concentrations. Each data point represents 16 wells from three separate plates. FKBP52 was measured using the output parameter of cytoplasmic or nuclear fluorescence or the difference between cytoplasm and nucleus average fluorescence intensity (cyt-nuc diff) with Compartmental Analysis Bioapplication. Values are normalized with the maximum control value.

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