Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-Zap-70 (Tyr319) Sandwich ELISA Kit #7171

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

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 Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

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

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.

Sensitivity

Sensitivity

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.

Background

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).

  1. Chu, D.H. et al. (1998) Immunol. Rev. 165, 167-180.
  2. Iwashima, M. et al. (1994) Science 263, 1136-1139.
  3. Neumeister, E.N. et al. (1995) Mol. Cell Biol. 15, 3171-3178.
  4. Chan, A.C. et al. (1995) EMBO J. 14, 2499-2508.
  5. Williams, B.L. et al. (1999) EMBO J. 18, 1832-1844.
  6. Di Bartolo, V. et al. (1999) J. Biol. Chem. 274, 6285-6294.
  7. Wiestner, A. et al. (2003) Blood 101, 4944-4951.
  8. Crespo, M. et al. (2003) N. Engl. J. Med. 348, 1764-1775.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Protocols

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

Products