Figure 1. Transfection of TPA differentiated THP-1 cells with Poly(dA:dT) stimulates phosphorylation of STING at Ser366 but does not affect the level of total STING. The relationship between lysate protein concentration from untransfected and Poly(dA:dT)-transfected differentiated THP-1 cells and the absorbance at 450 nm using the FastScan™ Phospho-STING (Ser366) ELISA Kit #82083 is shown in the upper figure. The corresponding western blots using STING antibody (left panel) and phospho-STING (Ser366) antibody (right panel) are shown in the lower figure. THP-1 cells were differentiated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (100 nM) for 18 hours, then transfected with Poly(dA:dT) (5 µg/mL) for 3 hours at 37°C, then lysed.
|Product Includes||Volume (with Count)||Solution Color|
|FastScan™ ELISA Microwell Strip Plate, 96 Well 53257||1 x 96 tests|
|STING Rabbit Capture mAb||1 x 1 ea||Green (Lyophilized)|
|Phospho-STING (Ser366) 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 #82083 Positive Control Type 2||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-STING (Ser366) ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of STING when phosphorylated at Ser366. 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-STING (Ser366) 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-STING (Ser366). Antibodies in kit are custom formulations specific to kit.
The FastScan™ Phospho-STING (Ser366) ELISA Kit detects endogenous levels of STING when phosphorylated at Ser366, 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.
Stimulator of interferon genes (STING, TMEM173, MITA) is a transmembrane adaptor protein that is a critical component of the cellular innate immune response to pathogenic cytoplasmic DNA (1,2). STING is a ubiquitously expressed protein found predominantly in the ER (1). The enzyme cGAMP synthase (cGAS) produces the second messenger cyclic-GMP-AMP (cGAMP) in response to cytoplasmic DNA (3,4). cGAMP binds and activates STING (3,4). In addition, detection of cytoplasmic DNA by nucleic acid sensors, including DDX41 or IFI16, results in STING activation (5,6). Following activation, STING translocates with TBK1 to perinuclear endosomes and gets phosphorylated by ULK1 at Ser366 (Ser365 in mouse) (7, 8). The TBK1 kinase phosphorylates and activates IRF-3 and NF-κB, which leads to the induction of type I interferon and other immune response genes (1,2,7).
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