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Phosphotyrosine anticorps

WB, IP, IF, IHC, ELISA, ICC Hôte: Lapin Polyclonal unconjugated
N° du produit ABIN361758
  • Antigène Tous les produits Phosphotyrosine
    Phosphotyrosine
    Reactivité
    Veuillez nous consulter SVP
    Hôte
    • 97
    • 27
    • 1
    Lapin
    Clonalité
    • 109
    • 16
    Polyclonal
    Conjugué
    • 44
    • 16
    • 12
    • 8
    • 4
    • 3
    • 3
    • 3
    • 2
    • 2
    • 2
    • 2
    • 2
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    • 2
    • 2
    • 1
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    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Cet anticorp Phosphotyrosine est non-conjugé
    Application
    • 88
    • 64
    • 41
    • 40
    • 39
    • 27
    • 25
    • 17
    • 13
    • 9
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    Western Blotting (WB), Immunoprecipitation (IP), Immunofluorescence (IF), Immunohistochemistry (IHC), ELISA, Immunocytochemistry (ICC)
    Specificité
    Detects proteins phosphorylated on tyrosine residues. Does not cross-react with phosphoserine or threonine.
    Purification
    Peptide Affinity Purified
    Immunogène
    Phosphotyrosine conjugated to KLH
  • Indications d'application
    • WB (1:500)
    • IHC (1:100)
    • ICC/IF (1:50)
    • ELISA (1:2000)
    • IP (1:100)
    • optimal dilutions for assays should be determined by the user.
    Commentaires

    A 1:250 dilution of ABIN361758 was sufficient for detection of tyrosine-phosphorylated species in mouse spleen lysates in western blot analysis.

    Restrictions
    For Research Use only
  • Validation #103692 (Proximity Ligation Assay)
    'Independent Validation' signe
    by
    Klinik für Anästhesie, Intensivmedizin und Schmerztherapie, Universitätsklinikum der Ruhr Universität Bochum
    No.
    #103692
    Date
    29.04.2019
    Antigène
    Phosphotyrosine
    Numéro du lot
    1508290
    Application validée
    Proximity Ligation Assay
    Contrôle positif

    U937 monocytic cells stimulated with ODN1826

    Contrôle négative

    Anti pY antibody (ABIN361758) alone

    Anti TLR9 antibody (ABIN5542492) alone

    Conclusion

    Passed. PLA signal visible in stimulated cells. Negative control with only one primary antibody shows no signal.

    'Independent Validation' signe
    Validation Images
    Protocole
    Anticorps primaire
    ABIN361758
    Anticorps secondaire
    ABIN5542492
    Full Protocol
    • Grow U937 cells in RPMI (Gibco, 21875-034) supplemented with 10% FCS (Gibco, A3840001) and Pen/Strep(Gibco, 15140-122), at 37°C and 5% CO2 in 500µL in 12-well plates.
    • Incubate the cells with 1µM ODN1826 for 4h.
    • Use cytospin to spin cells onto slide.
    • Fix cells on coverslide in 4% PFA for 20min at RT.
    • Wash cells 3x for 5min with PBS.
    • Permeabilize cells in PBS containing 0.1% Triton for 5min at RT.
    • Block non-specific binding with Duolink PLA Blocking Solution (Sigma-Aldrich) for 30min at RT.
    • Incubate cells with primary rabbit anti-pY antibody (antibodies-online, ABIN361758, lot 1508290) diluted 1:100 and primary mouse anti-TLR9 antibody (antibodies-online, ABIN5542492, lot 160127) diluted 1:200 ON at 4°C.
    • Wash cells 3x for 5min with PBS.
    • Incubate cells with Duolink In Situ PLA Probe anti-Rabbit MINUS (Sigma-Aldrich, DUO92005, lot SLBZ4516) and Duolink In Situ PLA Probe anti-Mouse mouse PLUS (Sigma-Aldrich, DUO92001, lot SLBZ8369) proximity probes according to manufacturers recommendations.
    • Wash cells 2x for 5min with PBS.
    • Perform ligation step according to the Duolink PLA kit (Sigma).
    • Perform amplification step according to the Duolink In Situ PlA Detection Kit Green (Sigma-Aldrich, DUO92014).
    • Mount coverslips on glass slides in ProLong Gold antifade reagent (Invitrogen, P36935, lot 1890418) containing DAPI.
    • Image acquisition with an Olympus widefield microscope.
    Notes

    We used double the amount the Phi29 polymerase for the amplifaction step than recommended.

  • Format
    Liquid
    Concentration
    0.25 mg/mL
    Buffer
    PBS pH 7.0, 50 % glycerol, 0.01 % sodium azide, Storage buffer may change when conjugated
    Agent conservateur
    Sodium azide
    Précaution d'utilisation
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Stock
    -20 °C
    Stockage commentaire
    -20°C
  • Antigène
    Phosphotyrosine
    Abstract
    Phosphotyrosine Produits
    Classe de substances
    Amino Acid
    Sujet
    Protein phosphorylation is an important posttranslational modification that serves many key functions to regulate a protein's activity, localization, and protein-protein interactions. Phosphorylation is catalyzed by various specific protein kinases, which involves removing a phosphate group from ATP and covalently attaching it to to a recipient protein that acts as a substrate. Most kinases act on both serine and threonine, others act on tyrosine, and a number (dual specificity kinases) act on all three. Because phosphorylation can occur at multiple sites on any given protein, it can therefore change the function or localization of that protein at any time (1). Changing the function of these proteins has been linked to a number of diseases, including cancer, diabetes, heart disease, inflammation and neurological disorders (2-4). In particular, the phosphorylation of tyrosine is considered one of the key steps in signal transduction and regulation of enzymatic activity (5). Phosphotyrosine can be detected through specific antibodies, and are helpful in facilitating the identification of tyrosine kinase substrates (6).
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