Product Pathways - PathScan ELISA
PathScan® Phospho-Zap-70 (Tyr319) Sandwich ELISA Kit #7171
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|Kit Includes||Volume||Solution Color|
|Zap-70 mAb Coated Microwells|
|P-Zap-70 Detection Ab||11 ml||Green|
|Anti-rabbit IgG, HRP-linked Antibody||11 ml||Red|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
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.
Specificity / Sensitivity
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.
ELISA - Western correlation
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.
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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- Iwashima, M. et al. (1994) Science 263, 1136-1139.
- Neumeister, E.N. et al. (1995) Mol. Cell Biol. 15, 3171-3178.
- Chan, A.C. et al. (1995) EMBO J. 14, 2499-2508.
- Williams, B.L. et al. (1999) EMBO J. 18, 1832-1844.
- Di Bartolo, V. et al. (1999) J. Biol. Chem. 274, 6285-6294.
- Wiestner, A. et al. (2003) Blood 101, 4944-4951.
- Crespo, M. et al. (2003) N. Engl. J. Med. 348, 1764-1775.
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- 7172 PathScan® Total Zap-70 Sandwich ELISA Kit
- 2701 Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody
- 2717 Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
For Research Use Only. Not For Use In Diagnostic Procedures.