Figure 1. TPA differentiated THP-1 cells transfected with Poly(dA:dT) stimulates phosphorylation of IRF-3 at Ser396 but does not affect the level of total IRF-3. The relationship between lysate protein concentration from untransfected and Poly(dA:dT)-transfected TPA differentiated THP-1 cells and the absorbance at 450 nm using the FastScan™ Phospho-IRF-3 (Ser396) ELISA Kit #50386 is shown in the upper figure. The corresponding western blots using IRF-3 antibody (left panel) and phospho-IRF-3 (Ser396) antibody (right panel) are shown in the lower figure. After overnight differentiation with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (100 nM), THP-1 cells were transfected with Poly(dA:dT) (5 µg/ml) for 3 hours at 37°C, and then lysed.
|Product Includes||Volume (with Count)||Solution Color|
|FastScan™ ELISA Microwell Strip Plate, 96 Well 53257||1 x 96 tests|
|Phospho-IRF-3 (Ser396) Rabbit Capture mAb||1 x 1 ea||Green (Lyophilized)|
|IRF-3 Mouse 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 #50386 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-IRF-3 (Ser396) ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IRF-3 when phosphorylated at Ser396. 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-IRF-3 (Ser396) 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-IRF-3 (Ser396). Antibodies in kit are custom formulations specific to kit.
IMPORTANT: This FastScan™ ELISA kit requires 4 washes at Step 6 of the protocol.
The FastScan™ Phospho-IRF-3 (Ser396) ELISA Kit detects endogenous levels of IRF-3 when phosphorylated at Ser396, 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.
Interferon regulatory factors (IRFs) comprise a family of transcription factors that function within the Jak/Stat pathway to regulate interferon (IFN) and IFN-inducible gene expression in response to viral infection (1). IRFs play an important role in pathogen defense, autoimmunity, lymphocyte development, cell growth, and susceptibility to transformation. The IRF family includes nine members: IRF-1, IRF-2, IRF-9/ISGF3γ, IRF-3, IRF-4 (Pip/LSIRF/ICSAT), IRF-5, IRF-6, IRF-7, and IRF-8/ICSBP. All IRF proteins share homology in their amino-terminal DNA-binding domains. IRF family members regulate transcription through interactions with proteins that share similar DNA-binding motifs, such as IFN-stimulated response elements (ISRE), IFN consensus sequences (ICS), and IFN regulatory elements (IRF-E) (2).
Explore pathways + proteins related to this product.