Phospho-p38 MAP Kinase (T180/Y182) Cell-Based ELISA
Phospho-p38 MAP Kinase (T180/Y182) Cell-Based ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
* Alternatively, this assay can be run over two days, which requires 2 hours 30 mins (day 1) and 4 hours (day 2).
R&D Systems Cell-Based ELISAs simultaneously measure the levels of two proteins in the same microplate well in whole, fixed cells, eliminating the need for lysate preparation. These simple, sensitive assays can be used with both adherent and non-adherent cells to analyze protein phosphorylation or assess total protein levels.
Cell-Based ELISA Kits are available in two formats: phospho-protein kits contain antibodies to measure both the phosphorylated and the total protein, while total protein kits contain antibodies to the protein of interest and a second housekeeping protein. These two proteins are simultaneously detected in the same microplate well, allowing signals derived from the target protein to be normalized to that of the second protein. This permits corrections for well-to-well variation and allows target protein levels to be accurately assessed and compared across multiple samples. This Phospho-
- No lysate preparation required
- Results with as few as 10,000 cells per well
- Measure two proteins simultaneously in the same well
- Ideal for measuring phosphorylation
- Design allows for normalization of well-to-well variations
- Complete kits require minimal optimization
- Suitable for high-throughput analysis of non-adherent cells
- Excellent alternative to Western blot and/or sandwich ELISA
- 96-well Microplate
- Primary antibody for target protein
- Primary antibody for normalization protein
- HRP-conjugated secondary antibody
- AP-conjugated secondary antibody
- HRP Substrate F1
- AP Substrate F2
- Blocking Buffer
- Wash Buffer
- Plate Sealers
OTHER REAGENTS REQUIRED
- 37% formaldehyde (Molecular Biology Grade; Sigma, Catalog # F8775), refer to MSDS prior to use
- 30% H2O2 (Sigma, Catalog # H1009). Refer to MSDS prior to use
- 1X PBS (Irvine Scientific, Catalog # 9240)
- Deionized or distilled water
- Pipettes and pipette tips
- Multi-channel pipette for washing
- Cell culture incubator
- Microfuge tubes
- Orbital shaker
- Fluorescence plate reader with two channels: excitation 540 nm/emission 600 nm and excitation 360 nm/emission 450 nm
Preparation and Storage
The p38 Mitogen-activated Protein Kinases (MAPKs) are a family of four related Ser/Thr kinases activated by proinflammatory cytokines and environmental stresses. All four p38 family members, alpha, beta, gamma, and delta, are phosphorylated by MKK3 and/or MKK6 at dual Thr and Tyr positions within the phosphoacceptor sequence Thr-Gly-Tyr. Once activated, p38 phosphorylates a number of targets, including the nuclear transcription factors ATF2 and Max.
The most frequently analyzed family member, p38 alpha, also known as SAPK2a and MAPK14, was initially purified as a kinase critical to the signaling cascade linking IL-1 to MAPKAPK-2 and the small heat shock protein HSP27. Ubiquitously expressed, p38 alpha is dually phosphorylated by MKK3 and MKK6 at Thr180 and Tyr182. Once activated, p38 alpha phosphorylates a number of targets, including the cytoplasmic kinases MNK 4 and PRAK5 and the nuclear transcription factors ATF2 1 and STAT1. Several promising compounds that inhibit p38 alpha are being investigated as potential therapies for arthritic and inflammatory diseases.
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