Recombinant
RabMAb

Recombinant Anti-ZAP70 (phospho Y292) antibody [EPR1073] (ab76501)

Overview

  • Product name

    Anti-ZAP70 (phospho Y292) antibody [EPR1073]
    See all ZAP70 primary antibodies
  • Description

    Rabbit monoclonal [EPR1073] to ZAP70 (phospho Y292)
  • Host species

    Rabbit
  • Specificity

    ab76501 only detects ZAP70 when phosphorylated at Tyrosine 292.
  • Tested applications

    Suitable for: WB, Flow Cyt, ICC/IF, Dot blotmore details
    Unsuitable for: IHC-P or IP
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human ZAP70 (phospho Y292). The exact sequence is proprietary.

  • Positive control

    • WB: Jurkat whole cell lysate (ab7899) treated with pervanadate. ICC/IF: Untreated and pervanadate treated Jurkat cells. Flow Cyt: Untreated and pervanadate treated Jurkat cells.
  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab76501 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
WB 1/5000 - 1/10000. Predicted molecular weight: 70 kDa.
Flow Cyt 1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF 1/250 - 1/500.
Dot blot 1/1000.
  • Application notes
    Is unsuitable for IHC-P or IP.
  • 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

    • All lanes : Anti-ZAP70 (phospho Y292) antibody [EPR1073] (ab76501) at 1/5000 dilution

      Lane 1 : Untreated Jurkat (human acute T cell leukemia) whole cell lysates
      Lane 2 : Jurkat (human acute T cell leukemia) treated with pervanadate whole cell lysates

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Peroxidase conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

      Predicted band size: 70 kDa
      Observed band size: 70 kDa


      Exposure time: 3 minutes


      Blocking and dilution buffer: 5% NFDM/TBST.

    • Dot blot analysis of varying amounts of ZAP70 (pY292) phospho peptide (Lane 1) and ZAP70 non-phospho peptide (Lane 2) using ab76501 at a dilution of 1/1000. A peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/2500).

      Exposure time: 3 minutes.

    • Immunocytochemistry/Immunofluorescence analysis of untreated and pervanadate treated Jurkat (human acute T cell leukemia) cells labelling ZAP70 (phospho Y292) with ab76501 at a dilution of 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Treated samples were starved overnight followed by incubation with 1mM pervanadate for 30 minutes. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. Negative control using secondary antibody only.

      Image shows increased cytoplasmic staining after pervanadate treatment.

    • Flow cytometric analysis of permeabilized Jurkat cells, untreated (green), pervanadate treated (red) stained with ab76501, at 1/20 dilution (blue) and pervanadate-treated Jurkat cells using the same antibody preincubated with phospho- ZAP70 (Tyr 292) peptide (blue) or non-phospho- ZAP70 (Tyr 292)).

    References

    ab76501 has not yet been referenced specifically in any publications.

    Customer reviews and Q&As

    1-2 of 2 Abreviews or Q&A

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Jurkat cell line)
    Loading amount
    40 µg
    Specification
    Jurkat cell line
    Treatment
    5mM H2O2 for 5min
    Gel Running Conditions
    Non-reduced Non-Denaturing (Native) (10% gel)
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 18°C

    Abcam user community

    Verified customer

    Submitted Dec 07 2012

    Answer

    Thank you very much for submitting your valuable reveiw of ab76501.
    I'm writing to follow-up with you, as the results were below average and we do guarantee the results of this antibody in Western blot with human samples. If the results are not satisfactory, we would be happy to offer you a replacement with another antibody such as ab12868, or alternatively a credit or refund.
    We do need some additional information in order to publish the review. Was the H2O2-treated Jurkat lysate the only sample run on the blot, or do some of the lanes show untreated lysate? If there are different samples on the blot, could you please indicate which sample was run in each lane? This can be added to the "additional notes" section of the review.
    We suggest using this antibody at a 1:5000-1:10,000 dilution in Western blot with ˜40 ug of protein, so using it at a 1:500 dilution with 40 ug of lysate could be causing the non-specific binding at 50 kDa. Have you tried a more dilute antibody and/or less lysate? If the specific band is weak at these conditions, perhaps treating the cells with H2O2 for a longer time period would be helpful.
    I look forward to hearing from you. If you have any questions or need anything from us, please let me know and I'll be happy to help.

    Read More

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

    Sign up