Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-p38α MAPK (Thr180/Tyr182) Sandwich ELISA Kit #7140

Kit Includes Volume Solution Color
p38alpha MAP Kinase Antibody Coated Microwells
Phospho-p38 MAPK Mouse mAb Detection Antibody 11 milliliters
Anti-mouse IgG HRP-Linked 11 milliliters
TMB Substrate 11 milliliters Colorless
STOP Solution 11 milliliters Colorless
Sealing Tape 2 sheets
20X Wash Buffer 25 milliliters Colorless
Sample Diluent 25 milliliters Blue
Cell Lysis Buffer (10X) # 9803 15 milliliters Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M R

Reactivity Key:  H=Human  M=Mouse  R=Rat

Description

CST's PathScan® Phospho-p38? MAP Kinase (Thr180/Tyr182) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-p38? MAP kinase (Thr180/Tyr182) protein. A p38? MAP Kinase Antibody* has been coated onto the microwells. After incubation with cell lysates, p38? MAP kinase protein is captured by the coated antibody. Following extensive washing, Phospho-p38 MAPK (Thr180/Tyr182) mouse mAb* is added to detect the captured phospho-p38? MAP kinase protein. HRP-linked Anti-mouse Antibody (#7076) is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of phospho-p38? MAP kinase (Thr180/Tyr182) protein.* Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Phospho-p38α MAP kinase (Thr180/Tyr182) Sandwich ELISA Kit detects endogenous levels of phospho-p38α MAP kinase (Thr180/Tyr182) protein. Using this Sandwich ELISA Kit #7140, a significant induction of phospho-p38α MAP kinase (Thr180/Tyr182) in NIH/3T3 cells treated with UV light is detected. However, levels of total p38α MAP kinase protein (phospho and nonphospho) remain unchanged, as shown by Western analysis using p38α MAP kinase Antibody #9218 (Figure 1). Both C6 and 293 cells treated either UV light or anisomycin show similar results (data not shown).

Western Blotting

Western Blotting

Figure 1: Treatment of NIH/3T3 cells with UV light stimulates phosphorylation of p38α MAP kinase at Thr180/Tyr182, detected by PathScan® Phospho-p38α MAP Kinase (Thr180/Tyr182) Sandwich ELISA Kit #7140 but does not affect the level of total p38α MAP kinase protein detected by p38α MAP Kinase Antibody #9218 via Western analysis. Absorbance at 450 nm is shown in the top figure, while the corresponding Western blot using Phospho-p38 MAP Kinase (Thr180/Tyr182) (28B10) Mouse mAb #9216 or p38α MAP Kinase Antibody #9218, is shown in the bottom figure.

Sandwich ELISA

Sandwich ELISA

Figure 2: The relationship between protein concentration of lysates from UV-treated NIH/3T3 cells and the absorbance at 450 nm is shown. NIH/3T3 cells (50-70% confluence) were treated with UV light and lysed after growth at 37oC for 30 min.

Background

p38 MAP kinase (MAPK), also called RK (1) or CSBP (2), is the mammalian orthologue of the yeast HOG kinase which participates in a signaling cascade controlling cellular responses to cytokines and stress (1-4). Four isoforms of p38 MAP kinase, p38α, β, γ (also known as ERK6 or SAPK3) and δ (also known as SAPK4) have been identified. Similar to the SAPK/JNK pathway, p38 MAP kinase is activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharides (LPS), UV light and growth factors (1-5). MKK3, MKK6 and SEK activate p38 MAP kinase by phosphorylation at Thr180 and Tyr182. Activated p38 MAP kinase has been shown to phosphorylate and activate MAPKAP kinase 2 (3) and to phosphorylate the transcription factors ATF-2 (5), Max (6) and MEF2 (5-8).

  1. Rouse, J. et al. (1994) Cell 78, 1027-1037.
  2. Han, J. et al. (1994) Science 265, 808-811.
  3. Lee, J.C. et al. (1994) Nature 372, 739-746.
  4. Freshney, N.W. et al. (1994) Cell 78, 1039-1049.
  5. Raingeaud, J. et al. (1995) J. Biol. Chem. 270, 7420-7426.
  6. Zervos, A.S. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 10531-10534.
  7. Zhao, M. et al. (1999) Mol. Cell. Biol. 19, 21-30.
  8. Yang, S.H. et al. (1999) Mol. Cell. Biol. 19, 4028-4038.
  9. Emre, Y. et al. (2007) Biochem J 402, 271-8.

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