The phospho-p38 (Thr180/Tyr182) kit enables the cell-based quantitative detection of phosphorylated p38 as a readout of the MAPK pathway.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • No-wash No-wash
The phospho-p38 (Thr180/Tyr182) kit enables the cell-based quantitative detection of phosphorylated p38 as a readout of the MAPK pathway.
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Overview

This HTRF cell based assay conveniently and accurately quantifies phosphorylated p-38-MAPK at Thr180/Tyr182. This kit has applications in inflammation, immune response, and oncology research. Using a streamlined protocol, amenable to low-volume formats, this kit can be used from basic research to High Throughput drug screening.

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Phospho-p38 MAPK (Thr180/Tyr182) assay principle

The Phospho-p38 MAPK (Thr180/Tyr182) assay measures p38 MAPK when phosphorylated at Thr180/Tyr182. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-p38 MAPK (Thr180/Tyr182) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
Phospho-p38 MAPK (Thr180/Tyr182) assay principle

Phospho-p38 MAPK (Thr180/Tyr182) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding phospho-p38 MAPK (Thr180/Tyr182) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-p38 MAPK (Thr180/Tyr182) 2-plate assay protocol

Phospho-p38 MAPK (Thr180/Tyr182) 1-plate assay protocol

Detection of Phosphorylated p38 MAPK (Thr180/Tyr182) with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Phospho-p38 MAPK (Thr180/Tyr182) 1-plate assay protocol

HTRF assay compared to WB using phospho-p38 MAPK cellular assay

HeLa cells were grown in a T175 flask 37°C, 5% Co2, 2 days. Stimulation was done with anisomycin 10µM for 15min. After elimination of cell culture medium, 3ml of supplemented lysis buffer were added and incubated for 30min. Soluble supernatants were collected after 10min centrifuging. Equal amounts of lysates were used for a side by side comparison of WB and HTRF. HTRF assay shows better sensitivity than Western Blot: 2500 cells for HTRF compared to10,000 cells for WB.
HTRF assay compared to WB using phospho-p38 MAPK cellular assay

Anisomycin dose-response on NIH 3T3 cells

Murine NIH 3T3 cells (50,000 cells/well) were stimulated for 45 minutes at 37°C with various concentrations of anisomycin. After a 30 minutes lysis incubation time, phosphorylated p38 MAPK was measured using the two-plate assay protocol.
Anisomycin dose-response on NIH 3T3 cells

Anisomycin dose-response on Hela cells

Various concentrations of HeLa cells (25, 50 and 100K cells/well) were incubated for 45 minutes at 37°C with various concentrations of anisomycin. After a 30 minutes lysis incubation time, phosphorylated p38 MAPK was measured using the two-plate assay protocol.
Anisomycin dose-response on Hela cells

Phospho-p38 MAPK (Thr180/Tyr182) simplified pathway

p38 MAPK is activated by a variety of cellular stresses, including osmotic shock, inflammatory cytokines, lipopolysaccharides (LPS), UltraViolet light, and growth factors. It is involved in cell differentiation and apoptosis. Activated p38 MAPK has been shown to phosphorylate and activate MAPKAP kinase 2 as well as phosphorylate the transcription factors ATF-2 , Max , and MEF2 . p38 MAPK plays a critical role in inflammation, immune response, apoptosis, cell differentiation, cell-cycle regulation and tumorigenesis.
Phospho-p38 MAPK signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Investigating kinase activity in a cellular context

HTRF cellular assays - Scientific Presentations

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

TCR signaling investigation with HTRF phospho assays

Study a pathway of interest in PBMC and T cells - Application Notes

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

An innate and adaptive immunity recap

Insight into the diversity of cells & signaling pathways - Guides

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

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