Phospho-p38 MAPK (Thr180/Tyr182) Cellular Assay Kit

HTRF® cellular assay kit for measuring phosphorylated p38 MAPK directly in cells

Based on our homogeneous and robust HTRF assays, the phospho-p38 MAPK kit is designed for detecting and studying activated p38 MAPK when phosphorylated at Thr180 and Tyr182 directly in whole cells. Using a streamlined protocol, amenable to low-volume format, this kit can be used from basic research to High Throughput drug screening.

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Assay Principle

The assay can be run with cell lysates or using whole cells.

Upon activation, p38 MAPK is phosphorylated on Thr180 and Tyr182 and after the lysis of the cell membrane, phospho-p38 MAPK can be detected using the kit reagents. The assays are based on sandwich immunoassays, each involving two monoclonal antibodies: the anti-phospho-p38 MAPK antibody labeled with Eu3+-cryptate and the anti-p38 MAPK antibody labeled with d2. These antibodies may be pre-mixed and added in a single dispensing step to further streamline the protocol.

The assays can be run under a two-plate protocol. They can also be further streamlined to a one-step assay.

Two-plate assay protocol

Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of phosphorylated p38 by HTRF reagents. This protocol enables the cells' viability and confluence to be monitored.

One-plate assay protocol

Detection of phosphorylated p38 with HTRF reagents is performed in a single plate used for plating, stimulation and lysis. No washing steps are required. This protocol, HTS designed, enables miniaturization while maintaining HTRF quality.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified pathway

p38 MAPK is activated by a variety of cellular stresses, including osmotic shock, inflammatory cytokines, lipopolysaccharides (LPS), UltraViolet light, and growth factors, and is involved in cell differentiation and apoptosis. Activated p38 MAPK has been shown to phosphorylate and activate MAPKAP kinase 2 and to 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.

Product Performance

1. Western Blot versus HTRF 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.

2. 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 minute lysis incubation time, phosphorylated p38 MAPK was measured using the two-plate assay protocol.

3. 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 minute lysis incubation time, phosphorylated p38 MAPK was measured using the two-plate assay protocol.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
P38 phospho-T180/Y182 kit 500 tests64P38PEG
P38 phospho-T180/Y182 kit 10,000 tests64P38PEH
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P38 phospho-T180/Y182 kit 1 x 96 tests64P38PET
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P38 phospho-T180/Y182 kit 50,000 tests64P38PEY
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Companion products

DescriptionCat. noProduct insertMSDS
Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF
P38 phospho-T180/Y182 kit control lysate64P38TDA
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Documents