Tools for Phosphorylation Research

p38 MAPK and Phospho-p38 MAPK (pThr¹⁸⁰/pTyr¹⁸²) ELISAS

P38 MAP kinase (MAPK, Mitogen-activated protein kinase), also known as a CDC-2-related protein kinase or CSBP (cytokine suppressive anti-inflammatory drug binding protein) ¹ regulates many cellular processes including inflammation, cell differentiation, cell growth and death. p38 MAPK is activated in response to a variety of extracellular stimuli including osmotic shock, cytokines, LPS (Prod. No. L 2654) and anisomycin (Prod. No. A 9789). The activation of p38 MAPK is mediated by several upstream kinases that phosphorylate p38 at threonine 180 and tyrosine 182. This dually phosphorylated form of p38 is a High Osmolarity Glycerol response kinase (HOG), a 43 kDa endogenous, active form of p38 a, b, and g required for growth under high osmolarity conditions. This form is present in the variety of cells following exposure to a broad range of extracellular stimuli, including UV B irradiation of human skin, endotoxins, osmotic and environmental stress, and, most recently, during ischemia/reperfusion.^2-5

Solid phase p38 MAPK ELISA (Prod. No. PM0100) and Phospho-p38 MAPK (pThr¹⁸⁰/pTyr¹⁸²) ELISA (Prod. No. CS0020) are designed for the in vitro quantitative detection of non-phopshorylated and dually phosphorylated human and mouse p38 MAPK in cell lysates. A monoclonal capture antibody specific for p38 MAPK (regardless of phosphorylation state) has been coated onto a 96 multiwell plate. p38 MAPK antigen in samples and standards binds to the capture antibody. A detection antibody specific for the phosphorylated or non-phosphorylated p38 MAPK is added and binds to the immobilized p38 MAPK protein. An anti-rabbit IgG-HRP completes the four-member sandwich. The reaction is visualized by a TMB substrate and the intensity of the color is directly proportional to the concentration of p38 MAPK present in the original sample. The reaction is read at 450 nm in a multiwell plate reader. The assays detect both native and recombinant p38 MAPK and phospho-p38 MAPK (pThr¹⁸⁰/pTyr¹⁸²) proteins. The assays are highly sensitive (below 16 pg/mL and below 0.8 units/mL, respectively), specific, fast (4 hours total time) and simple (offer precoated plates, all incubations proceed at room temperature and the reagents are supplied ready to use).

Size:   96 wells

Performance Characteristics:

• Precision
     • Low intra-assay variability from 3.9% to 7.3%
     • Low inter-assay variability from 5.8% to 9.7%
• Linearity of dilution – correlation coefficient for various dilutions was 0.99 for both assays
• Recovery – average recovery was 93-97%

Storage:   Store at 2 to 8 °C

Components:

• Standards: p38 MAPK (Prod. No. P 8870) and Phospho-p38 MAPK (pThr¹⁸⁰/pTyr¹⁸²) (Prod. No. P 7995) - lyophilized recombinant p38 MAPK and recombinant p38 MAPK dually phosphorylated by MKK6.
• Standard/Sample diluent buffer
• Monoclonal anti-p38 MAPK-coated 96 multiwell plate
• Anti-p38 MAPK (Prod. No. P 7620) or Anti-phospho-p38 MAPK (pThr¹⁸⁰/pTyr¹⁸²) (Prod. No. P 7870)
• Anti-rabbit IgG-HRP concentrate 100X
• HRP Diluent
• Wash Buffer Concentrate 25X
• Stabilized TMB Chromogen
• Stop Solution
• Plate Sealers

References:

1. Han, J., et al, Biochem. Biophys. Acta., 1265, 224-227 (2000).
2. Bulavin, D.V., et al., Nature, 411, 102-107 (2001).
3. Pfundt, R., et al., J. Pathol., 193, 248-255 (2001).
4. Weinbrenner, C., et al., J. Mol. Cell Cardiol., 29, 2383-2391 (1997).
5. Ge, B., et al., Science, 295, 1291-1294 (2002).

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