p38 MAP Kinase Assay

p38 MAPK Assay for High Content Analysis

Millipore’s HCS231 p38 MAP Kinase Assay provides a complete solution for identifying and quantifying the phosphorylation state of endogenous cellular p38 MAPK at Thr180/Tyr182 via cellular imaging. The reagents in the kit have been specifically optimized for HCS applications. The assay is designed to enable visualization and quantitative detection of phosphorylated p38, facilitating the identification and characterization of activators and inhibitors of p38. Key applications of this assay include p38 inhibitor screening, characterization of cellular stress and inflammatory responses, and in vitro toxicology applications. The nuclear dye (Hoechst 33342) may be used for measurements of cell number, DNA content and nuclear size. Additionally, the assay can be multiplexed with other probes for analysis of proximal cellular events, e.g., for upstream or downstream signaling pathway profiling or for simultaneous screening for cytotoxicity.

p38 mitogen-activated protein kinases (p38 MAPKs) convert extracellular signals into specific cellular responses through interacting with and phosphorylating downstream targets. p38 MAPK are regulated by a wide variety of stimuli, often associated with stress, such as pro-inflammatory cytokines like IL-1 and TNFα, UV radiation, reactive oxygen species, osmotic shock, chemical toxins such as anisomycin, and biological agents such as bacterial lipopolysaccharide (LPS) and viruses. Upon stimulation, p38 MAPK are dually phosphorylated on their common TGY motif (Thr180/Tyr182 in human p38α), resulting in their activation and translocation from the cytoplasm to the nucleus. Once activated, p38 is able to phosphorylate a wide array of targets, including numerous kinases such as MK2, MK3, PRAK, MSK, and MNK. p38 also phosphorylates many transcription factors, including STAT1, p53, NFAT, ATF-2, and MEF2. Pharmacological inhibitors of p38 have entered Phase II clinical trials for inflammatory conditions such as rheumatoid arthritis, neuropathic pain, atherosclerosis, psoriasis, myelodysplastic syndromes and chronic obstructive pulmonary diseases.

Biochemical recombinant kinase assays and tumor necrosis factor alpha (TNFα) secretion assays are typically used to evaluate p38 inhibitors, but these assays do not provide insight into subcellular localization or proximal intracellular events. Since phospho-specific antibody detection of phosphorylation at Thr180/Tyr182 provides a well-established measure of cellular p38 activation or inhibition, High Content Screening technology represents a particularly useful tool for cell-based analysis of p38 MAPK.

• High Content Analysis
• Immunofluorescence
• Immunocytochemistry
• Kinase Assay

Cell-based kinase assay, cell signaling, cellular stress, drug discovery

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

• High-Content Screening
• High-Content Analysis

Sufficient reagents for 5 X 96 well plates

• 1. Rabbit Anti-Phospho-p38 (Thr180/Tyr182) HCS Primary Antibody, 100X
• 2. HCS Secondary Antibody (donkey anti-rabbit IgG, Cy3 conjugate), 200X
• 3. Hoechst HCS Nuclear Stain, 200X
• 4. HCS Fixation Solution with Phenol Red, 2X
• 5. HCS Immunofluorescence Buffer, 1X
• 6. HCS Wash Buffer, 1X
• 7. Anisomycin, 2.5 mM in DMSO, 250X
• 8. SB 202190, 2.5mM in DMSO, 250X
• 9. DMSO for Compound Serial Dilution
• 10. Compound Dilution Buffer
• 11. Plate Sealers

Store kit components under the conditions indicated on the labels. Discard any remaining reagents after 6 months.

Fluorescent

1. Sterile, tissue culture-treated black/clear bottom microplates suitable for High-Content Imaging.

2. Cell-type for assay, e.g., HeLa (human cervical adenocarcinoma, ATCC #CCL-2), A549 (human lung carcinoma, ATCC #CCL-185) or HepG2 (human hepatocellular carcinoma, ATCC #HB-8065).

3. Tissue culture instruments/supplies (including 37ºC incubator, growth media, flasks/plates, detachment buffer, etc.) for cell type of interest.

4. HCS imaging/analysis system, e.g., GE Healthcare IN Cell Analyzer 1000 with Investigator software. System must be equipped with beam-splitters and filters capable of reading emission spectra in the blue and red ranges.