Figure 1. Treatment of Jurkat cells with H2O2 stimulates phosphorylation of Zap-70 at Tyr319 but does not affect the level of total Zap-70. The relationship between lysate protein concentration from untreated and H2O2-treated Jurkat cells and the absorbance at 450 nm using the FastScan™ Total Zap-70 ELISA Kit #23925 is shown in the upper figure. The corresponding western blots using Zap-70 antibody (left panel) and phospho-Zap-70 (Tyr319) antibody (right panel) are shown in the lower figure. After serum starvation, Jurkat cells were treated with 11 mM H2O2 for 3 minutes 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|
|Zap-70 Rabbit Capture mAb||1 x 1 ea||Green (Lyophilized)|
|Zap-70 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|
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™ Total Zap-70 ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Zap-70. 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 Zap-70 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 Zap-70. Antibodies in kit are custom formulations specific to kit.
The FastScan™ Total Zap-70 ELISA Kit detects endogenous levels of Zap-70 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.Species Reactivity:
The Syk family protein tyrosine kinase Zap-70 is expressed in T and NK cells and plays a critical role in mediating T cell activation in response to T cell receptor (TCR) engagement (1). Following TCR engagement, Zap-70 is rapidly phosphorylated on several tyrosine residues through autophosphorylation and transphosphorylation by the Src family tyrosine kinase Lck (2-6). Tyrosine phosphorylation correlates with increased Zap-70 kinase activity and downstream signaling events. Expression of Zap-70 is correlated with disease progression and survival in patients with chronic lymphocytic leukemia (7,8).
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. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
U.S. Patents 9,086,407, 9,261,500, and 9,476,874, foreign equivalents, and child patents deriving therefrom.