Phospho-ZAP70/Syk (Tyr319, Tyr352) Monoclonal Antibody (n3kobu5), PerCP-eFluor 710

货号：46-9006-41,46-9006-42

规格：25 Tests,100 Tests

价格：1442,3711

产品类型：流式抗体

品牌：eBioscience

物种：人/小鼠

宿主：小鼠

抗体亚型：IgG2b, kappa

克隆号：n3kobu5

荧光染料：PerCP-eFluor™ 710

类型:	单抗	同型对照：	Mouse IgG2b kappa Isotype Control (eBMG2b), PerCP-eFluor 710
浓度：	5 µL/Test	用法：	5 µL (0.03 µg)/test(Flow)

产品详细信息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. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.Staining Protocol: All protocols work well for this monoclonal antibody. Use of Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins allows for the greatest flexibility for detection of surface and intracellular (cytoplasmic) proteins. Use of Protocol B: One-step protocol: intracellular (nuclear) proteins is recommended for staining of transcription factors in conjunction with surface and phosphorylated intracellular (cytoplasmic) proteins. Protocol C: Two-step protocol: Fixation/Methanol allows for the greatest discrimination of phospho-specific signaling between unstimulated and stimulated samples, but with limitations on the ability to stain specific surface proteins (refer to "Clone Performance Following Fixation/Permeabilization" located in the Best Protocols Section under the Resources tab online). All Protocols can be found in the Flow Cytometry Protocols: "Staining Intracellular Antigens for Flow Cytometry Protocol" located in the Best Protocols Section under the Resources tab online.PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light.Fixation: Samples can be stored in IC Fixation Buffer (cat. 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (cat. 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser.Filtration: 0.2 µm post-manufacturing filtered.靶标信息ZAP70, a 70 kDa member of the Syk tyrosine kinase family, plays a central role in lymphocyte activation and development, and is implicated in several immune disorders. 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 292terminates the transient activation of ZAP70 and attentuates lymphocyte signaling.

数据

Phospho-ZAP70/Syk (Tyr319, Tyr352) Antibody (46-9006-42) in FlowIntracellular staining of normal human peripheral blood cells unstimulated (orange histogram) or stimulated with hydrogen peroxide-activated sodium pervanadate for 5 minutes (purple histogram) with Anti-Human/Mouse phospho-ZAP-70/SYK (Y319/Y352) PerCP-eFluor® 710. The Intracellular Fixation and Permeabilization Buffer SetProduct # 88-8824-00) and protocol were used for staining. Lymphocytes in the CD3+ gate were used for analysis.

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