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RecombinantRabMAb

Recombinant Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)

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Western blot - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)
  • Western blot - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)
  • Flow Cytometry (Intracellular) - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)
  • Immunoprecipitation - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)
  • Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)

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 - BSA and Azide free
  • Suitable for: IHC-P, Flow Cyt (Intra), WB, IP, ICC/IF
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 647 HRP Unconjugated

Overview

  • Product name

    Anti-ZAP70 antibody [YE291] - BSA and Azide free
    See all ZAP70 primary antibodies

  • Description

    Rabbit monoclonal [YE291] to ZAP70 - BSA and Azide free

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IHC-P, Flow Cyt (Intra), WB, IP, ICC/IFmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Jurkat cell lysate. IHC-P: Human lymph node tissue. Flow Cyt (intra): Jurkat cells IP: Jurkat lysate.

  • General notes

    ab247258 is the carrier-free version of ab32429.

    Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

    This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.

    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
    For more information see here.

    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

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab247258 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
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt (Intra)
Use at an assay dependent concentration.
WB
Use at an assay dependent concentration. Detects a band of approximately 70 kDa (predicted molecular weight: 70 kDa).
IP
Use at an assay dependent concentration.
ICC/IF
Use at an assay dependent concentration.
Notes
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt (Intra)
Use at an assay dependent concentration.
WB
Use at an assay dependent concentration. Detects a band of approximately 70 kDa (predicted molecular weight: 70 kDa).
IP
Use at an assay dependent concentration.
ICC/IF
Use at an assay dependent concentration.

Target

  • 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
    modifications

    Phosphorylated 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

  • Database links

    • Alternative names

      • 70 kDa zeta associated protein antibody
      • 70 kDa zeta-associated protein antibody
      • EC 2.7.10.2 antibody
      • FLJ17670 antibody
      • FLJ17679 antibody
      • Selective T cell defect antibody
      • SRK antibody
      • STD antibody
      • Syk related tyrosine kinase antibody
      • Syk-related tyrosine kinase antibody
      • Truncated ZAP kinase antibody
      • Tyrosine protein kinase ZAP70 antibody
      • Tyrosine-protein kinase ZAP-70 antibody
      • TZK antibody
      • ZAP 70 antibody
      • ZAP70 antibody
      • ZAP70_HUMAN antibody
      • Zeta chain associated protein kinase 70kD antibody
      • Zeta chain associated protein kinase 70kDa antibody
      • Zeta chain associated protein kinase 70kDa isoform 1 antibody
      • Zeta chain associated protein kinase 70kDa isoform 2 antibody
      • Zeta chain of T cell receptor associated protein kinase 70 antibody
      • Zeta chain TCR associated protein kinase 70kD antibody
      • Zeta chain TCR associated protein kinase 70kDa antibody
      see all

    Images

    • Western blot - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)
      Western blot - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)
      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 kDa



      False 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.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32429). 

    • Western blot - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)
      Western blot - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)
      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 kDa



      Anti-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)

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32429). 

    • Flow Cytometry (Intracellular) - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)
      Flow Cytometry (Intracellular) - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)

      Intracellular Flow Cytometry analysis of Jurkat cells labeling ZAP70 with ab32429 at 1/80 dilution (red) or rabbit IgG as negative control (green). 

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32429). 

       

       

       

    • Immunoprecipitation - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)
      Immunoprecipitation - Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)

      This data was developed using ab32429, the same antibody clone in a different buffer formulation.

      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: abab32429 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.

    • Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)
      Anti-ZAP70 antibody [YE291] - BSA and Azide free (ab247258)

    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.

    Click here to view the general protocols

    References (0)

    Publishing research using ab247258? Please let us know so that we can cite the reference in this datasheet.

    ab247258 has not yet been referenced specifically in any publications.

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