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Figure 1. Treatment of ACHN cells with Human Interleukin-4 (hIL-4) stimulates phosphorylation of Stat6 at Tyr641 but does not effect the level of total Stat6. The relationship between lysate protein concentration from untreated and hIL-4-treated ACHN cells and the absorbance at 450 nm using the FastScan™ Phospho-Stat6 (Tyr641) ELISA Kit #58967 is shown in the upper figure. The corresponding western blots using phospho-Stat6 (Tyr641) antibody (left panel) and Stat6 antibody (right panel) are shown in the lower figure. After serum starvation, ACHN cells were treated with 100 ng/ml hIL-4 #8919 for 15 minutes at 37°C and then lysed.Learn more about how we get our images
|Product Includes||Volume (with Count)||Solution Color|
|FastScan™ ELISA Microwell Strip Plate, 96 Well 53257||1 x 96 tests|
|Stat6 Rabbit Capture mAb||1 x 1 ea||Green (Lyophilized)|
|Phospho-Stat6 (Tyr641) Rabbit HRP-linked mAb||1 x 1 ea||Red (Lyophilized)|
|FastScan™ ELISA Capture Antibody Diluent||1 x 3 ml||Green|
|FastScan™ ELISA HRP Antibody Diluent||1 x 3 ml|
|TMB Substrate 7004||1 x 11 ml|
|STOP Solution 7002||1 x 11 ml|
|Sealing Tape||1 x 1 ea|
|ELISA Wash Buffer (20X) 9801||1 x 25 ml|
|FastScan™ ELISA Cell Extraction Buffer (5X) 69905||1 x 10 ml|
|FastScan™ ELISA Cell Extraction Enhancer Solution (50X) 25243||1 x 1 ml|
|FastScan™ ELISA Kit #58967 Positive Control||2 x 1 ea|
NOTE: Prepare solutions with deionized/purified water or equivalent.
Prepare only as much reagent as needed on the day of the experiment.
*IMPORTANT: The provided FastScan™ ELISA Cell Extraction Enhancer Solution (50X) may precipitate when stored at 4°C. To dissolve, warm briefly at 37°C and mix gently. The FastScan™ ELISA Cell Extraction Enhancer Solution (50X) can be stored at room temperature to avoid precipitation.
NOTE: The 1X Cell Extraction Buffer contains phosphatase inhibitors. Protease inhibitors should be added to the 1X Cell Extraction Buffer immediately prior to lysing cells. Additional phosphatase inhibitors can also be added (e.g. Protease/Phosphatase Inhibitor Cocktail (100X) #5872, not supplied).
For adherent cells
For suspension cells
NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.
*NOTE: Certain FastScan™ ELISA Kits may require additional washes at this step. Any requirements for additional washes will be specifically noted in the product “Description” of the kit’s datasheet.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted May 2018
revised November 2018
The FastScan™ Phospho-Stat6 (Tyr641) ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Stat6 when phosphorylated at Tyr641. To perform the assay, sample is incubated with a capture antibody conjugated with a proprietary tag and a second detection antibody linked to HRP, forming a sandwich with phospho-Stat6 (Tyr641) in solution. This entire complex is immobilized to the plate via an anti-tag antibody. The wells are then washed to remove unbound material. TMB is then added. The magnitude of observed signal is proportional to the quantity of phospho-Stat6 (Tyr641). Antibodies in kit are custom formulations specific to kit.
The FastScan™ Phospho-Stat6 (Tyr641) ELISA Kit detects endogenous levels of Stat6 when phosphorylated at Tyr641 as shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. FastScan™ ELISA is a trademark of Cell Signaling Technology, Inc. U.S. Patents 9,086,407, 9,261,500, and 9,476,874, foreign equivalents, and child patents deriving therefrom.
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|58967C||1 Kit (96 assays)|
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