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.
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.
|Product Includes||Volume||Solution Color|
|P-Stat6 (Tyr641) Rabbit mAb Coated Microwell||96 tests|
|Stat6 Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
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.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
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.Species Reactivity:
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).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.