Cell Signaling Technology

Product Pathways - Jak/Stat Pathway

PathScan® Phospho-Stat6 (Tyr641) Sandwich ELISA Kit #7275

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
Stat6 Mouse mAb coated microwells 96 tests
Phospho-Stat6 (Tyr641) Rabbit Detection Antibody 11 ml Green
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml 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, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Phospho-Stat6 (Tyr641) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Stat6 protein phosphorylated at Tyr641. A Stat6 Mouse mAb has been coated onto the microwells. After incubation with cell lysates, Stat6 (phospho or nonphospho) protein is captured by the coated antibody. Following extensive washing, Phospho-Stat6 (Tyr641) Rabbit Detection Antibody is added to detect the captured phospho-Stat6 protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Stat6 phosphorylated at Tyr641.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-Stat6 (Tyr641) Sandwich ELISA Kit detects endogenous levels of phospho-Stat6 (Tyr641) in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of ACHN cells with Human Interleukin-4 (hIL-4) #8919 stimulates phosphorylation of Stat6 at Tyr641, as detected by PathScan® Phospho-Stat6 (Tyr641) Sandwich ELISA Kit #7275, but does not affect levels of total Stat6 protein detected by PathScan® Total Stat6 Sandwich ELISA Kit #7267. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Stat6 Antibody #9362 (left panel) or Phospho-Stat6 (Tyr641) (C11A12) Rabbit mAb #9364 (right panel) are shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2. Relationship between protein concentration of lysates from ACHN cells, untreated or treated with Human Interleukin-4 (hIL-4) #8919, and the absorbance at 450 nm is shown. Cells (80% confluence) were treated with hIL-4 (100 ng/ml) and lysed after incubation at 37ºC for 15-20 minutes.

Background

Upon activation by Janus kinases, Stat6 translocates to the nucleus where it regulates cytokine-induced gene expression. Stat6 is activated via phosphorylation at Tyr641 and is required for responsiveness to IL-4 and IL-13 (1-4). In addition, Stat6 is activated by IFN-α in B cells, where it forms transcriptionally active complexes with Stat2 and p48 (5,6). Protein phosphatase 2A is also involved in regulation of IL-4-mediated Stat6 signaling (7).

  1. Nelms, K. et al. (1999) Ann. Rev. Immunol. 17, 701-738.
  2. Malabarba, M.G. et al. (1996) Biochem. J. 319, 865-872.
  3. Hou, J. et al. (1994) Science 265, 1701-1706.
  4. Quelle, F.W. et al. (1995) Mol. Cell. Biol. 15, 3336-3343.
  5. Takeda, K. et al. (1996) Nature 380, 627-630.
  6. Gupta, S. et al. (1999) J. Immunol. 163, 3834-3841.
  7. Woetmann, A. et al. (2003) J. Biol. Chem. 278, 2787-2791.

Application References

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