Recombinant Anti-ZAP70 antibody [YE291] (ab32429)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [YE291] to ZAP70
- Suitable for: WB, IHC-P, IP, Flow Cyt (Intra)
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-ZAP70 antibody [YE291]
See all ZAP70 primary antibodies -
Description
Rabbit monoclonal [YE291] to ZAP70 -
Host species
Rabbit -
Tested applications
Suitable for: WB, IHC-P, IP, Flow Cyt (Intra)more details -
Species reactivity
Reacts with: Human -
Immunogen
Synthetic peptide within Human ZAP70 aa 550 to the C-terminus (C terminal). The exact sequence is proprietary.
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Positive control
- WB: Jurkat cell lysate. IHC-P: Human lymph node tissue. Flow Cyt (intra): Jurkat cells IP: Jurkat lysate.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
YE291 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Isotype control
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32429 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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WB |
1/500. Detects a band of approximately 70 kDa (predicted molecular weight: 70 kDa).
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IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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IP |
1/50.
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Flow Cyt (Intra) |
1/80.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
Notes |
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WB
1/500. Detects a band of approximately 70 kDa (predicted molecular weight: 70 kDa). |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
IP
1/50. |
Flow Cyt (Intra)
1/80. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
Target
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Function
Plays a role in T-cell development and lymphocyte activation. Essential for TCR-mediated IL-2 production. Isoform 1 induces TCR-mediated signal transduction, isoform 2 does not. -
Tissue specificity
Expressed in T- and natural killer cells. -
Involvement in disease
Defects in ZAP70 are the cause of selective T-cell defect (STD) [MIM:176947]. STD is an autosomal recessive form of severe combined immunodeficiency characterized by a selective absence of CD8-type T-cells. -
Sequence similarities
Belongs to the protein kinase superfamily. Tyr protein kinase family. SYK/ZAP-70 subfamily.
Contains 1 protein kinase domain.
Contains 2 SH2 domains. -
Domain
The SH2 domains bind to the phosphorylated tyrosine-based activation motif (TAM) of CD3Z and the non-canonical phosphorylated tyrosine-based activation motif (TAM) of RHOH. -
Post-translational
modificationsPhosphorylated on tyrosine residues upon T-cell antigen receptor (TCR) stimulation. Tyr-319 phosphorylation is essential for full activity. -
Cellular localization
Cytoplasm. Cell membrane. After antigen stimulation, isoform 1 concentrates at the immunological synapse and isoform 2 remains cytoplasmic. Co-localizes together with RHOH in the immunological synapse. RHOH is required for its proper localization to the cell membrane and cytoskeleton fractions in the thymocytes. - Information by UniProt
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Database links
- Entrez Gene: 7535 Human
- Omim: 176947 Human
- SwissProt: P43403 Human
- Unigene: 234569 Human
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Alternative names
- 70 kDa zeta associated protein antibody
- 70 kDa zeta-associated protein antibody
- EC 2.7.10.2 antibody
see all
Images
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All lanes : Anti-ZAP70 antibody [YE291] (ab32429) at 1/500 dilution
Lane 1 : Wild-type Jurkat cell lysate
Lane 2 : ZAP70 CRISPR-Cas9 edited Jurkat cell lysate
Lane 3 : MOLT-4 cell lysate
Lane 4 : Raji cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 70 kDa
Observed band size: 70 kDaFalse colour image of Western blot: Anti-ZAP70 antibody [YE291] staining at 1/500 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32429 was shown to bind specifically to ZAP70. A band was observed at 70 kDa in wild-type Jurkat cell lysates with no signal observed at this size in ZAP70 CRISPR-Cas9 edited cell line ab273841 (CRISPR-Cas9 edited cell lysate ab273795). The band observed in the CRISPR-Cas9 edited lysate lane below 70 kDa is likely to represent a truncated form of ZAP70. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and ZAP70 CRISPR-Cas9 edited Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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ZAP70 was immunoprecipitated from 0.35 mg Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10 µg with ab32429 at 1/50 dilution (2µg). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10 µg
Lane 2: ab32429 IP in Jurkat whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab32429 in Jurkat whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
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All lanes : Anti-ZAP70 antibody [YE291] (ab32429) at 1/1000 dilution
Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
Lane 2 : Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed)
Predicted band size: 70 kDaAnti-GAPDH antibody, ab8245 (1/20000) was used as a primary antibody for the loading control and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed, ab216776 (1/10000) was used as a loading control secondary antibody.
Lanes 1-2: Merged signal (red and green). Green – ab32429 observed at 70 kDa. Red - loading control ab8245 observed at 36 kDa.
ab32429 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.The expression profile observed in Raji is consistent with the literature (PMID: 25275600).
Negative control: Raji (PMID: 25275600)
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Ab32429, at a 1/100 dilution, staining ZAP70 in paraffin embedded human lymph node tissue by immunohistochemistry.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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Anti-ZAP70 antibody [YE291] (ab32429) at 1/500 dilution + Jurkat cell lysate
Predicted band size: 70 kDa
Observed band size: 70 kDa -
Intracellular Intracellular Intracellular Flow Cytometry analysis of Jurkat cells labeling ZAP70 with ab32429 at 1/80 dilution (red) or rabbit IgG as negative control (green).
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ab32429 showing positive staining in Normal tonsil tissue.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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ab32429 showing negative staining in Normal liver tissue.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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ab32429 showing negative staining in Normal heart tissue.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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ab32429 showing negative staining in Normal kidney tissue.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (4)
ab32429 has been referenced in 4 publications.
- Ren L et al. AQP9 and ZAP70 as immune-related prognostic biomarkers suppress proliferation, migration and invasion of laryngeal cancer cells. BMC Cancer 22:465 (2022). PubMed: 35477402
- Mäkinen A et al. Expression of BCL6 in paediatric B-cell acute lymphoblastic leukaemia and association with prognosis. Pathology 53:875-882 (2021). PubMed: 34049715
- Li Y et al. Malignant ascite-derived extracellular vesicles inhibit T cell activity by upregulating Siglec-10 expression. Cancer Manag Res 11:7123-7134 (2019). PubMed: 31534365
- Miyata-Takata T et al. Expression of T-cell receptor signalling pathway components in extranodal NK/T-cell lymphoma. Histopathology 73:1030-1038 (2018). PubMed: 30102799