Figure 1. Treatment of 3T3 cells with UV irradiation stimulates phosphorylation of c-Jun at Ser63 but does not affect the level of total c-Jun. The relationship between lysate protein concentration from untreated and UV-treated 3T3 cells and the absorbance at 450 nm using the FastScan™ Phospho-c-Jun (Ser63) ELISA Kit #32614 is shown in the upper figure. The corresponding western blots using phospho-c-Jun (Ser63) antibody (left panel) and c-Jun antibody (right panel) are shown in the lower figure. 3T3 cells were treated with UV (50 mJ/cm2 with 60 min recovery at 37°C) and then lysed.
|REACTIVITY||H M R|
|Product Includes||Volume (with Count)||Solution Color|
|FastScan™ ELISA Microwell Strip Plate, 96 Well 53257||1 x 96 tests|
|Phospho-c-Jun (Ser63) Rabbit Capture mAb||1 x 1 ea||Green (Lyophilized)|
|c-Jun 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 #32614 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
Protocol Id: 1704
The FastScan™ Phospho-c-Jun (Ser63) ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of c-Jun when phosphorylated at Ser63. 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-c-Jun (Ser63) 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-c-Jun (Ser63). Antibodies in kit are custom formulations specific to kit.
The FastScan™ Phospho-c-Jun (Ser63) ELISA Kit detects endogenous levels of c-Jun when phosphorylated at Ser63 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
c-Jun is a member of the Jun family containing c-Jun, JunB, and JunD, and is a component of the transcription factor activator protein-1 (AP-1). AP-1 is composed of dimers of Fos, Jun, and ATF family members and binds to and activates transcription at TRE/AP-1 elements (reviewed in 1). Extracellular signals including growth factors, chemokines, and stress activate AP-1-dependent transcription. The transcriptional activity of c-Jun is regulated by phosphorylation at Ser63 and Ser73 through SAPK/JNK (reviewed in 2). Knock-out studies in mice have shown that c-Jun is essential for embryogenesis (3), and subsequent studies have demonstrated roles for c-Jun in various tissues and developmental processes including axon regeneration (4), liver regeneration (5), and T cell development (6). AP-1 regulated genes exert diverse biological functions including cell proliferation, differentiation, and apoptosis, as well as transformation, invasion and metastasis, depending on cell type and context (7-9). Other target genes regulate survival, as well as hypoxia and angiogenesis (8,10). Research studies have implicated c-Jun as a promising therapeutic target for cancer, vascular remodeling, acute inflammation, and rheumatoid arthritis (11,12).
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.