Invitrogen™ Phospho-ZAP70/Syk (Tyr319, Tyr352) Monoclonal Antibody (n3kobu5), PE, eBioscience™, Invitrogen™

Catalog No.	Quantity
50-112-9140	100 Tests
50-112-8993	25 Tests

Catalog No. 50-112-9140 Supplier Invitrogen™ Supplier No. 12900642

Description

Mouse Monoclonal Antibody

Description: This n3kobu5 monoclonal antibody recognizes human and mouse zeta chain-associated protein of 70 kD (also known as ZAP-70) and spleen tyrosine kinase (also known as SYK) when phosphorylated on Y319 and Y352, respectively. ZAP-70 and SYK are members of the SYK protein tyrosine kinase (PTK) family that are phosphorylated and activated by Src family PTKs. ZAP-70/SYK Y319/Y352 are located in the so-called interdomain of ZAP-70/SYK (between the N-terminal dual SH2 domains and the C-terminal kinase domain). Phosphorylation of ZAP-70 Y319 by Lck is necessary for T cell receptor (TCR)-dependent association of ZAP-70 with Lck and phospholipase C gamma and subsequent activation of calcium-dependent and Ras signaling cascades. SYK Y352 phosphorylation by Fyn/Lyn is critical for propagation of B cell receptor (BCR) signaling and for B cell development. Specificity of this n3kobu5 clone was determined by ELISA, flow cytometry, and western blotting. Applications Reported: This n3kobu5 antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This n3kobu5 antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of stimulated normal human peripheral blood cells. This can be used at 5 μL (0.03 μg) per test. A test is defined as the amount (μg) of antibody that will stain a cell sample in a final volume of 100 μL.

ZAP-70 is a cytosolic protein tyrosine kinase that is a member of the Syk family of proteins. ZAP-70 is expressed exclusively in T-cells and natural killer cells and is required for T-cell receptor activation. Upon T cell antigen receptor (TCR) engagement, ZAP70 is phosphorylated on tyrosines 292, 315 and 319 in the interdomain B, located between the SH2 and kinase domains. Phosphorylation of both tyrosines 315 (a Vav binding site) and 319 (a Lck binding site) enhances ZAP70 function in mediating lymphocyte signaling, while tyrosine 292 terminates the transient activation of ZAP70 and attentuates lymphocyte signaling.

Specifications

• Antigen: Phospho-ZAP70/Syk (Tyr319, Tyr352)
• Applications: Flow Cytometry
• Classification: Monoclonal
• Clone: n3kobu5
• Concentration: 5 μL/Test
• Conjugate: PE
• Formulation: PBS with BSA and 0.09% sodium azide; pH 7.2
• Gene: ZAP70
• Gene Accession No.: P43403, P43404
• Gene Alias: 70 kDa zeta-associated protein; 70 kDa zeta-chain associated protein; ADMIO2; EC 2.7.10.2; FLJ17670; FLJ17679; I79_004087; IMD48; kinase ZAP70; mrtle; mur; Selective T cell defect; Srk; STCD; STD; syk-related protein tyrosine kinase; syk-related tyrosine kinase; Tyrosine-protein kinase ZAP-70; Tyrosine-protein kinase ZAP-70;TZK; TZK; ZA70; Zap70; ZAP-70; Zeta chain associated protein kinase 70kDa; zeta chain of T cell receptor associated protein kinase; zeta chain of T cell receptor associated protein kinase 70; zeta chain of T cell receptor associated protein kinase 70kDa; zeta-chain (TCR) associated protein kinase; zeta-chain (TCR) associated protein kinase 70; zeta-chain (TCR) associated protein kinase 70kDa; zeta-chain associated protein kinase 70kDa; zeta-chain associated protein kinase, 70kD
• Gene Symbols: ZAP70
• Host Species: Mouse
• Purification Method: Affinity chromatography
• Quantity: 100 Tests
• Regulatory Status: RUO
• Primary or Secondary: Primary
• Gene ID (Entrez): 22637, 7535
• Target Species: Human, Mouse
• Content And Storage: 4°C, store in dark, DO NOT FREEZE!
• Product Type: Antibody
• Form: Liquid
• Isotype: IgG2b κ

Frequently Asked Questions (FAQs)

How does eBioscience determine the specificity of their phosphorylation-specific antibodies?

Antibodies are tested and validated in a variety of ways. First, we verify that in a panel of cells in which different pathways have been induced, we see phosphorylation specific staining only in the cells in which the specific pathway of interest has been activated. Second, we verify that phosphorylation specific staining is observed only in cell types in which the protein is expressed and not in cell types in which the protein is not expressed. Third, whenever possible, western immunoblotting is used to confirm the presence of a band(s) of the appropriate size(s) in stimulated/treated cells (and not in unstimulated/untreated cells, as appropriate).

How long can cells that have been fixed and placed in methanol be stored for use with eBioscience antibodies? What is the recommended storage temperature? How about storage of fixed cells in eBioscience fixation buffers?

We have data demonstrating that fixed cells stored in methanol are stable at -20 degrees C or at -80 degrees C for several weeks. We do not recommend storing fixed cells in eBioscience Foxp3/Transcription Factor Buffer. However, cells fixed with eBioscience Foxp3/Transcription Factor Buffer or eBioscience Intracellular (IC) fixation buffer can be stored in eBioscience IC fixation buffer for up to 3 days at 4 degrees C in the dark.
Note: Please be aware that higher compensation values may be seen with tandem dyes if any other fixative is used apart from eBioscience IC fixation buffer, for 3-day storage.

What is the optimal protocol for eBioscience phosphorylation-specific antibodies?

Each antibody has been tested in three different buffer systems: IC fixation and permeabilization, Foxp3/Transcription Factor Buffer, and IC fixation Buffer/Methanol. The recommended buffer system(s) will be noted on the Technical Data Sheet for the specific antibody.

What is the best way to maintain surface staining when using the methanol-based protocol?

Because methanol can destroy the epitope recognized by some antibodies, ideally surface staining would be performed at the beginning of the protocol with a methanol-resistant fluorochrome (like eFluor 450, FITC, and eFluor 660).
Please recognize that surface staining before stimulation can have undesired effects due to activation of the cell caused by antibody binding. We recommend staining after stimulation/treatment, fixation, and methanol treatment.

Can I use other buffer systems from other companies when using eBioscience phosphorylation-specific antibodies?

In general, you can use other companies' buffer systems provided that their buffers are of a similar composition as the buffers recommended. Be aware that results will vary depending upon the buffer system used. eBioscience antibodies have been optimized for use in eBioscience buffers and we have not tested all of our antibodies in other buffer systems. Thus, we highly recommend using our optimized protocol and buffer systems.

What are the appropriate controls to run when using eBioscience phosphorylation-specific antibodies?

It is important to understand whether the stimulation/treatment results in an upregulation or a down regulation of the phosphorylation event. Thus, it is recommended that changes in phosphorylation levels in stimulated/treated versus unstimulated/untreated cells be compared. A histogram or density plot overlay these two treatments will provide a better assessment of a change in phosphorylation event than using isotype controls.

Can I perform phosphorylation specific staining and stain for intracellular cytokines at the same time when using eBioscience phosphorylation-specific antibodies?

In theory, if the buffer systems are compatible for the antibodies of interest, this can be done. In practice, cytokine translation and phosphorylation events do not typically occur in the same timeframe, therefore, it may be difficult to detect both events in the same sample. The results will be depended on the kinetics and should be determined for each system and protein of interest.

When using eBioscience phosphorylation-specific antibodies, can I perform phosphorylation specific staining together with transcription factor staining at the same time? If so, what buffer and protocol do I use?

In theory, this can be done if the antibodies against both the transcription factor and the phosphorylated protein work in the same buffer system. For example, Anti-Human/Mouse phsopho-H2AX and all transcription factor eBioscience antibodies offered will work in the Foxp3/Transcription Factor Buffer System (Cat. No. 00-5523-00).

For eBioscience phosphorylation-specific antibodies, can total and phosphorylated protein be analyzed in parallel?

In theory, this can be done if the antibodies to both proteins work in the same buffer system and if the antibodies recognize different epitopes. Please refer to the individual Technical Data Sheets for information about buffer compatibility. Also, consider using eBioscience InstantOne ELISA Kits for immunoassay quantitation of both total and phosphorylated proteins such as AKT1/2/3, JNK1/2/3, NFκB, STAT3, and STAT5.

For Research Use Only.