Figure 1: Treatment of Jurkat cells with hydrogen peroxide stimulates phosphorylation of Zap-70 at Tyr319, detected by PathScan® Phospho-Zap-70 (Tyr319) Sandwich ELISA kit #7171, but does not affect the level of total Zap-70 detected by PathScan® Total Zap-70 Sandwich ELISA kit #7172. OD 450 readings are shown in the top figure, while the corresponding Western blot using Phospho-Zap-70 (Tyr319) Antibody #2701 (right panel) or Zap-70 Antibody #2705 (left panel), is shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and hydrogen peroxide treated Jurkat cells and kit assay optical density readings. Jurkat cells (0.8 x 106 cells/ml) were treated with hydrogen peroxide (2 mM) for 2 min at 25oC, and then lysed.
|Product Includes||Volume||Solution Color|
|Zap-70 Mouse mAb Coated Microwells||96 tests|
|Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml|
|HRP Diluent||11 ml|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
CST's PathScan® Phospho-Zap-70 (Tyr319) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-Zap-70 (Tyr319) protein. A Zap-70 Mouse mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-Zap-70 proteins are captured by the coated antibody. Following extensive washing, Phospho-Zap-70 (Tyr319) Ab is added to detect the captured phospho-Zap-70 (Tyr319) protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-Zap-70 (Tyr319) protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Phospho-Zap-70 (Tyr319) Sandwich ELISA Kit detects endogenous levels of phospho-Zap-70 (Tyr319) enzyme. As shown in Figure 1, using this Sandwich ELISA Kit #7171, a significant induction of Phospho-Zap-70 (Tyr319) in Jurkat cells treated with hydrogen peroxide is detected. However, the level of total Zap-70 (phospho and non-phospho), detected by the Total Zap-70 Sandwich ELISA Kit #7172, remains unchanged. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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).
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