Figure 1. Treatment of A-431 cells with EGF stimulates phosphorylation of Stat3 at Ser727 but does not effect the level of total Stat3. The relationship between lysate protein concentration from untreated and EGF-treated A-431 cells and the absorbance at 450 nm using the FastScan™ Phospho-Stat3 (Ser727) ELISA Kit #16242 is shown in the upper figure. The corresponding western blots using phospho-Stat3 (Ser727) antibody (left panel) and Stat3 antibody (right panel) are shown in the lower figure. After serum starvation, A-431 cells were either left untreated or treated with 100 ng/mL EGF for 5 minutes at 37°C, then lysed.
|REACTIVITY||H M R Mk|
|Product Includes||Volume (with Count)||Solution Color|
|FastScan™ ELISA Microwell Strip Plate, 96 Well 53257||1 x 96 tests|
|Stat3 Rabbit Capture mAb||1 x 1 ea||Green (Lyophilized)|
|Phospho-Stat3 (Ser727) 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 #16242 Positive Control Type 1||1 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).
NOTE: A select number of FastScan™ ELISA kits do not contain a positive control, please refer to Product Includes table on the datasheet for a list of included reagents. Should you need support on how to generate a positive control for those kits, contact CST technical support at [email protected].
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 2019
Protocol Id: 1924
The FastScan™ Phospho-Stat3 (Ser727) ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Stat3 when phosphorylated at Ser727. 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-Stat3 (Ser727) 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-Stat3 (Ser727). Antibodies in kit are custom formulations specific to kit.
The FastScan™ Phospho-Stat3 (Ser727) ELISA Kit detects endogenous levels of Stat3 when phosphorylated at Ser727 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.
Human, Mouse, Rat, Monkey
The Stat3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors (1) and is required for murine fetal development (2). Research studies have shown that Stat3 is constitutively activated in a number of human tumors (3,4) and possesses oncogenic potential (5) and anti-apoptotic activities (3). Stat3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding (6,7). Transcriptional activation seems to be regulated by phosphorylation at Ser727 through the MAPK or mTOR pathways (8,9). Stat3 isoform expression appears to reflect biological function as the relative expression levels of Stat3α (86 kDa) and Stat3β (79 kDa) depend on cell type, ligand exposure, or cell maturation stage (10). It is notable that Stat3β lacks the serine phosphorylation site within the carboxy-terminal transcriptional activation domain (8).
Explore pathways + proteins related to this product.
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.