Antibody data
- Antibody Data
- Antigen structure
- References [1]
- Comments [0]
- Validations
- Flow cytometry [1]
- Other assay [1]
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- Product number
- 46-9006-41 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-ZAP70/Syk (Tyr319, Tyr352) Monoclonal Antibody (n3kobu5), PerCP-eFluor™ 710, eBioscience™
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- 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 (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 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.
- Reactivity
- Human, Mouse
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- n3kobu5
- Vial size
- 25 Tests
- Concentration
- 5 µL/Test
- Storage
- 4° C, store in dark, DO NOT FREEZE!
Submitted references CD19-CAR-T Cells Bearing a KIR/PD-1-Based Inhibitory CAR Eradicate CD19(+)HLA-C1(-) Malignant B Cells While Sparing CD19(+)HLA-C1(+) Healthy B Cells.
Tao L, Farooq MA, Gao Y, Zhang L, Niu C, Ajmal I, Zhou Y, He C, Zhao G, Yao J, Liu M, Jiang W
Cancers 2020 Sep 13;12(9)
Cancers 2020 Sep 13;12(9)
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Intracellular 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 & Permeabilization Buffer Set Product # 88-8824-00) and protocol were used for staining. Lymphocytes in the CD3+ gate were used for analysis.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 2 Dephosphorylating P-Zap70 by iKP CAR via intracellular PD-1 domain. ( A ) Flow cytometric analysis of CD19 and HLA-C1 expression in Daudi cells or Raji cells by using APC-anti-human CD19 and PE-anti-human HLA-C antibodies. ( B ) Expression analysis of P-Zap70 in different CAR-T cells by flow cytometry. iKP-19-CAR-T/iKPt-19-CAR-T cells and CD19-CAR-T cells were exposed to Daudi cells or Raji cells for 6 h at a 1:1 ratio in RPMI-1640 medium, stained with PE-anti-human P-Zap70 antibody and MFI of P-Zap70 was statistically analyzed ( n = 4 different donors). All the experiments were conducted in triplicate manner using PBMCs from each donor. *** p < 0.001. Error bars represent +- SD. The CD19 CAR positive rate was unified using UT cells in all the co-culture experiments in this study.