Anti-p-ZAP-70 (Tyr319) Antibody, clone 1B10 ZooMAb® Rabbit Monoclonal
SIGMA/ZRB1717 - recombinant, expressed in HEK 293 cells
Synonym: Tyrosine-protein kinase ZAP-
Product Type: Chemical
Immunohistochemistry (Paraffin) Enhanced Validation - Recombinant Antibody Technology Formalin Fixed Paraffin Embedded (FFPE) Human Tonsil (A) and Negative control (no primary) (B) tissue sections were prepared using heat-induced epitope retrieval (HIER). Immunostaining was performed using a 1:100 dilution of Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal. Reactivity was detected using a Goat Anti-Rabbit IgG and HRP-DAB. Cytoplasmic/membranous staining was observed in lymphatic nodules & epithelium of Human Tonsil tissue sections.
Immunohistochemistry (Paraffin) Enhanced Validation - Recombinant Antibody Technology Formalin Fixed Paraffin Embedded (FFPE) Human Tonsil (A) and Negative control (no primary) (B) tissue sections were prepared using heat-induced epitope retrieval (HIER). Immunostaining was performed using a 1:100 dilution of Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal. Reactivity was detected using a Goat Anti-Rabbit IgG and HRP-DAB. Cytoplasmic/membranous staining was observed in lymphatic nodules & epithelium of Human Tonsil tissue sections.
Immunohistochemistry (Paraffin) Enhanced Validation - Recombinant Antibody Technology Formalin Fixed Paraffin Embedded (FFPE) Human Tonsil (A) and Negative control (no primary) (B) tissue sections were prepared using heat-induced epitope retrieval (HIER). Immunostaining was performed using a 1:100 dilution of Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal. Reactivity was detected using a Goat Anti-Rabbit IgG and HRP-DAB. Cytoplasmic/membranous staining was observed in lymphatic nodules & epithelium of Human Tonsil tissue sections.
Immunocytochemistry Enhanced Validation - Recombinant Antibody Technology Immunofluorescent analysis of untreated Jurkat cells (A and B) and Jurkat cells treated with H2O2 (C and D) cell line was performed using a 1:100 dilution of Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal and visualized with a Goat Anti-Rabbit secondary antibody conjugated to Alexa Fluor® 488 (Green). Actin filaments have been labeled with phalloidin (Red). Nucleus is stained with DAPI (Blue). This antibody positively stains the membrane and cytoplasm.
Immunocytochemistry Enhanced Validation - Recombinant Antibody Technology Immunofluorescent analysis of untreated Jurkat cells (A and B) and Jurkat cells treated with H2O2 (C and D) cell line was performed using a 1:100 dilution of Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal and visualized with a Goat Anti-Rabbit secondary antibody conjugated to Alexa Fluor® 488 (Green). Actin filaments have been labeled with phalloidin (Red). Nucleus is stained with DAPI (Blue). This antibody positively stains the membrane and cytoplasm.
Immunocytochemistry Enhanced Validation - Recombinant Antibody Technology Immunofluorescent analysis of untreated Jurkat cells (A and B) and Jurkat cells treated with H2O2 (C and D) cell line was performed using a 1:100 dilution of Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal and visualized with a Goat Anti-Rabbit secondary antibody conjugated to Alexa Fluor® 488 (Green). Actin filaments have been labeled with phalloidin (Red). Nucleus is stained with DAPI (Blue). This antibody positively stains the membrane and cytoplasm.
Immunocytochemistry Enhanced Validation - Recombinant Antibody Technology Immunofluorescent analysis of untreated Jurkat cells (A and B) and Jurkat cells treated with H2O2 (C and D) cell line was performed using a 1:100 dilution of Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal and visualized with a Goat Anti-Rabbit secondary antibody conjugated to Alexa Fluor® 488 (Green). Actin filaments have been labeled with phalloidin (Red). Nucleus is stained with DAPI (Blue). This antibody positively stains the membrane and cytoplasm.
Immunocytochemistry Enhanced Validation - Recombinant Antibody Technology Immunofluorescent analysis of untreated Jurkat cells (A and B) and Jurkat cells treated with H2O2 (C and D) cell line was performed using a 1:100 dilution of Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal and visualized with a Goat Anti-Rabbit secondary antibody conjugated to Alexa Fluor® 488 (Green). Actin filaments have been labeled with phalloidin (Red). Nucleus is stained with DAPI (Blue). This antibody positively stains the membrane and cytoplasm.
Western Blotting Enhanced Validation - Recombinant Antibody Technology Lysates from untreated Jurkat cells (Lane 1) and Jurkat cells treated with H2O2 (Lane 2) were probed with Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal (1:1,000 dilution). Proteins were visualized using a Donkey Anti-Rabbit IgG secondary antibody conjugated to HRP and a chemiluminescence detection system. Arrow indicates p-ZAP-70 (Tyr319) (~70 kDa).
Flow Cytometry Enhanced Validation - Recombinant Antibody Technology Staining of one million untreated Jurkat cells (Grey histogram) and H2O2 treated Jurkat cells (Orange histogram) was performed using 0.1 μg of Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal (Orange histogram) followed by a PE-conjugated Donkey Anti-Rabbit IgG secondary antibody.
Flow Cytometry Enhanced Validation - Recombinant Antibody Technology Staining of one million Jurkat cells treated with H2O2 was performed using 0.1 μg of Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal (Yellow histogram) or the equivalent amount of a Rabbit IgG isotype control (Grey histogram), followed by a PE-conjugated Donkey Anti-Rabbit IgG secondary antibody.
Peptide Inhibition Assay Enhanced Validation - Recombinant Antibody Technology Lysates from Jurkat cells treated with H2O2 were probed with Cat. No. ZRB1717, Anti-p-ZAP-70 (Tyr319), clone 1B10 ZooMAb® Rabbit Monoclonal (1:1,000 dilution). Proteins were visualized using a Donkey Anti-Rabbit IgG secondary antibody conjugated to HRP and a chemiluminescence detection system. Immobilon® Signal Enhancer was used. Arrow indicates p-ZAP-70 (Tyr319) (~70 kDa). Lanes 1: Incubated without any peptide (Control). 2: Incubated with phosphorylated Zap-70 (Tyr319) peptide. 3: Incubated with non- phosphorylated Zap-70 (Tyr319) peptide.
12 Principles of Green Chemistry: Principle 1—Waste Prevention. This product has a smaller environmental footprint through reduction of waste in the manufacturing process.
12 Principles of Green Chemistry: Principle 4—Designing Safer Chemicals
Chemical products should be designed to affect their desired function while minimizing their toxicity.
Chemical products should be designed to affect their desired function while minimizing their toxicity.
12 Principles of Green Chemistry: Principle 6—Design for Energy Efficiency. This product was re-engineered to utilize less energy in the manufacturing process.
| antibody form | purified antibody |
| antibody product type | primary antibodies |
| biological source | rabbit |
| clone | 1B10, recombinant monoclonal |
| conjugate | unconjugated |
| description | recombinant, expressed in HEK 293 cells |
| enhanced validation | recombinant expression Learn more about Antibody Enhanced Validation  |
| epitope sequence | C-terminus |
| form | lyophilized |
| greener alternative category | Aligned |
| greener alternative product characteristics | Waste Prevention Designing Safer Chemicals Design for Energy Efficiency Learn more about the Principles of Green Chemistry . |
| isotype | IgG |
| mol wt | calculated mol wt 69.87 kDa |
| observed mol wt ~70 kDa | |
| packaging | antibody small pack of 25 μL |
| product line | ZooMAb® learn more |
| Protein ID accession no. | NP_001070  |
| purified by | using Protein A |
| Quality Level | 200 |
| recombinant | expressed in HEK 293 cells |
| shipped in | ambient |
| species reactivity | human |
| species reactivity (predicted by homology) | mouse, rat, bovine, monkey |
| storage temp. | 2-8°C |
| technique(s) | flow cytometry: suitable |
| immunocytochemistry: suitable | |
| immunohistochemistry (formalin-fixed, paraffin-embedded sections): suitable | |
| peptide synthesis: suitable | |
| western blot: suitable | |
| UniProt accession no. | P43403 |
| Application: | Quality Control Testing Evaluated by Western Blotting in lysate from Jurkat cells treated with hydrogen peroxide (H2O2). Western Blotting Analysis (WB): A 1:1,000 dilution of this antibody detected phospho-ZAP-70 (Tyr319) in lysate from Jurkat cells treated with hydrogen peroxide (H2O2; 10 mM; 3 min.), but not in untreated cells. Tested applications Flow Cytometry Analysis: 0.1 μg from a representative lot detected p-ZAP-70 (Tyr319) in one million Jurkat cells treated with H2O2. Immunocytochemistry Analysis: A 1:100 dilution from a representative lot detected p-ZAP-70 (Tyr319) in Jurkat cells treated with H2O2. Immunohistochemistry (Paraffin) Analysis: A 1:100 dilution from a representative lot detected p-ZAP-70 (Tyr319) in human tonsil tissue sections. Peptide Inhibition Assay:: Target band detection in lysate from Jurkat cells treated with H2O2 was prevented by preblocking of a representative lot with phosphorylated Zap-70 (Tyr319) peptide, but not the corresponding non-phosphopeptide. Note: Actual optimal working dilutions must be determined by end user as specimens, and experimental conditions may vary with the end user |
| Disclaimer: | Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals. |
| General description: | We are committed to bringing you greener alternative products, which adhere to one or more of The 12 Principles of Green Chemistry.This antibody is Preservative-free, produced without the harm or sacrifice of animals and exceptionally stable to allow for ambient shipping and storage if needed and thus aligns with "Waste Prevention", "Designing Safer Chemicals" and "Design for Energy Efficiency". Click here for more information. |
| General description: | ZooMAb® antibodies represent an entirely new generation of recombinant monoclonal antibodies. Each ZooMAb® antibody is manufactured using our proprietary recombinant expression system, purified to homogeneity, and precisely dispensed to produce robust and highly reproducible lot-to-lot consistency. Only top-performing clones are released for use by researchers. Each antibody is validated for high specificity and affinity across multiple applications, including its most commonly used application. ZooMAb® antibodies are reliably available and ready to ship when you need them. |
| Immunogen: | KLH-conjugated linear peptide corresponding to 10 amino acids surrounding phosphotyrosine 319 from the internal region of human ZAP70. |
| Legal Information: | ZooMAb is a registered trademark of Merck KGaA, Darmstadt, Germany |
| Physical form: | Purified recombinant rabbit monoclonal antibody IgG, lyophilized in PBS, 5% Trehalose, normal appearance a coarse or translucent resin. The PBS/trehalose components in the ZooMAb formulation can have the appearance of a semi-solid (bead like gel) after lyophilization. This is a normal phenomenon. Please follow the recommended reconstitution procedure in the data sheet to dissolve the semi-solid, bead-like, gel-appearing material. The resulting antibody solution is completely stable and functional as proven by full functional testing. Contains no biocide or preservatives, such as azide, or any animal by-products. Larger pack sizes provided as multiples of 25 μL. |
| Reconstitution: | 30 μg/mL after reconstitution at 25 μL per vial. Please refer to guidance on suggested starting dilutions and/or titers per application and sample type. |
| Specificity: | Clone 1B10 is a ZooMAb® Rabbit recombinant monoclonal antibody that specifically detects ZAP-70 phosphorylated on tyrosine 319. |
| Storage and Stability: | Recommend storage of lyophilized product at 2-8°C; Before reconstitution, micro-centrifuge vials briefly to spin down material to bottom of the vial; Reconstitute each vial by adding 25 μL of filtered lab grade water or PBS; Reconstituted antibodies can be stored at 2-8°C, or -20°C for long term storage. Avoid repeated freeze-thaws. |
| Target description: | Tyrosine-protein kinase ZAP-70 (UniProt: P43403; also known as EC:2.7.10.2, 70 kDa zeta-chain associated protein, Syk-related tyrosine kinase) is encoded by the ZAP70 (also known as SRK) gene (Gene ID: 7535) in human. ZAP-70 is a tyrosine kinase that is expressed in T cells and NK cells and plays an essential role in regulation of the adaptive immune response. Its expression is also observed in early thymocytes and pro/pre-B-cells. It is involved in regulating motility, adhesion, and cytokine expression of mature T-cells and in thymocyte development. In quiescent T cells, it is cytoplasmic and upon TCR activation, it is recruited at the plasma membrane by interacting with CD247/CD3Z. ZAP-70 contains two tandem SH2 domains (aa 10-102 and 163-254) that bind to the doubly phosphorylated tyrosine-based activation motif (ITAM) of CD247/CD3Z and the non-canonical phosphorylated tyrosine-based activation motif (TAM) of Ras Homolog Family Member H (RHOH). Its protein kinase domain is localized in amino acids 338-600. It has an interdomain B region (aa 255-337), located between the second SH2 and the kinase domain, contains 3 tyrosine residues (Tyr-292, 315, 319) that are phosphorylated following TCR activation. Phosphorylation of Tyr-315 and Tyr-319 is reported to be are essential for ZAP70 positive function on T-lymphocyte activation whereas Tyr-292 is shown to have a negative regulatory role. Within the protein kinase domain, Tyr-492 and Tyr-493 are also shown to be phosphorylated after TCR induction. Here Tyr-492 plays a negative regulatory role and Tyr-493 has a positive role and can be dephosphorylated by Protein Tyrosine Phosphatase Non-Receptor Type 22 (PTN22). Aberrant high expression of ZAP-70 has been reported in a subset of Chronic Lymphocytic Leukemia (CLL) subjects and reflects an unfavorable clinical outcome. This ZooMAb® recombinant monoclonal antibody, generated by our propriety technology, offers significantly enhanced specificity, affinity, reproducibility, and stability over conventional monoclonals. (Ref.: Chen, J., et al.(2020). Front. Oncol. 10; 595832; Fischer, A., et al. (2010). Semin. Immunopathol. 32(2); 107-116; Chan, AC., et al. (1992). Cell. 71(4); 649-662). |
| Storage Temp. | 2-8°C |
| UNSPSC | 12352203 |