Heme Oxygenase 1 and Phospho-p38 Activation Kit

The kit contains a rabbit polyclonal antibody that detects only phosphorylated p38, a mouse monoclonal antibody for heme oxygenase 1, DyLight™-conjugated Secondary Antibodies and various other reagents and buffers required for immunofluorescence detection.

Heme oxygenase 1 is a microsomal enzyme (32 kDa) that catalyzes the oxidation of heme to antioxidants, biliveridin and carbon monoxide and protects cells from wide variety of stress conditions through activation of p38 mitogen activated protein kinase (MAPK). Carbon monoxide produced during oxidation of heme by heme oxygenase 1 activates p38 kinase, which confers tissue protection through inhibition of cytokine production. Phosphorylation at Thr180/Tyr182 activates p38 kinase. Phosphorylated p38 translocates to nucleus where it phosphorylates and activates several substrates involved in cell stress, inflammation, growth and differentiation. Heme oxygenase 1 and p38 is activated by stressors including oxidative stress, hypoxia, heat shock, heavy metals and cytokines.

In normal cells, heme oxygenase 1 protein levels are low and p38 kinase is inactivated (i.e., non-phosphorylated); however, when cells are treated with copper sulfate, heme oxygenase 1 is induced and p38 is phosphorylated. Heme oxygenase 1 induction increases protein staining in the nucleus and cytoplasm. Phospho-p38 translocates to nucleus and is measured by the average nuclear intensity or difference between nuclear and cytoplasmic intensities. The assay was developed in A549 cells and optimized using the Thermo Scientific ArrayScan HCS Reader and Compartmental Analysis Bioapplication Software Module. Cells stained using this kit also can be imaged using fluorescence or confocal microscopy.

Figure 2. Staining of phospho-p38 and heme oxygenase 1 in A549 cells treated with or without copper sulfate (0.5 mM) for 24 hours. Cells were stained according to the kit protocol and imaged using the ArrayScan HCS Reader. Copper sulfate treatment leads to activation of p38 and heme oxygenase 1 and increased nuclear staining.

Figure 3. Dose response curves of phospho-p38 and heme oxygenase 1 in A549 cells. The average nuclear intensities for phospho-p38 and heme oxygenase 1 are plotted. Data represents mean ± SD from three plates (eight wells per 96-well plate per dose of copper sulfate).