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Ga16 anticorps

Reactivité: Humain IP, WB Hôte: Lapin Polyclonal unconjugated
N° du produit ABIN110590
  • Antigène
    Ga16
    Reactivité
    Humain
    Hôte
    Lapin
    Clonalité
    Polyclonal
    Application
    Immunoprecipitation (IP), Western Blotting (WB)
  • Restrictions
    For Research Use only
  • Validation #100205 (Western Blotting)
    'Independent Validation' signe
    by
    Laboratorio Universitario di Ricerca Medica, Univsersità degli Studi di Verona
    No.
    #100205
    Date
    27.11.2016
    Antigène
    Ga16
    Numéro du lot
    82100
    Application validée
    Western Blotting
    Contrôle positif
    QGP1 and PT45 lysates
    Contrôle négative
    MIA PaCa-2 cells lysate; PT45 shRNA knock-down cell lysate
    Conclusion
    'Independent Validation' signe
    Validation Images
    Protocole
    Anticorps primaire
    ABIN110590
    Anticorps secondaire
    goat anti-rabbit IgG HRP antibody (Sigma-Aldrich, A0545)
    Full Protocol
    • Pancreatic cancer cells lines QGP1, PT45, and MIA PaCa-2 were grown in DMEM 10% FBS at 37°C in 5% CO2.
    • PT45 cells were transduced with lentiviral particles to express shRNA that specifically targets G alpha-15 mRNA.
    • Wash cells with PBS.
    • Collect and lyse cells in RIPA buffer supplemented with protease inhibitor cocktail.
    • Determine total protein content of the lysates using bicinchoninic acid (BCA) method.
    • Adjust samples to 1µg/µl protein using RIPA buffer and Laemmli buffer.
    • Boil samples for 5min and separate 20µg of each sample on a 10% acrylamide gel at 100V until the salt/dye front reached the bottom.
    • Transfer proteins onto PVDF membrane.
    • Block the membrane with TBST containing 5% milk for 1 h at room temperature.
    • Incubation with primary GNA15 antibody (antibodies-online, ABIN110590, lot 82100) diluted 1:500 in TBST 5% milk for 2h at RT.
    • Wash membrane 4x 5min in TBST.
    • Incubation with secondary goat anti-rabbit IgG HRP antibody (Sigma-Aldrich, A0545) 1:10000.
    • Wash membrane 4x 5 minutes in TBST.
    • Reveal protein bands using Luminata Forte Western HRP substrate (Millipore, WBLUF0500) as substrate and Syngene G-box to image light emission.
    • Strip membranes (2% SDS, 62.5mM TrisHCl pH6.8, 0.8% beta-mercaptoethanol) and subsequently incubate with a beta-actin loading control antibody (Santa Cruz clone (C4), sc-47778, lot D0108) diluted 1:1000 and secondary anti-mouse IgG HRP antibody (Sigma-Aldrich, A2554) diluted 1:10000.
    • Wash and reveal the protein as described above.
      Notes
      Passed. The inhibition proves that the antibody is specific for GNA15 and it does not recognize one of the more abundant and ubiquitous homologs, GNAQ or GNA11.
    • Format
      Lyophilized
      Stock
      4 °C
    • Antigène
      Ga16
      Sujet
      Ga 16 is the only hetero-trimeric G proteins with a restricted expression pattern in hematopoietic cells. Differentiation of promyelocyticcells leads to decreased expression of Ga 16. This G protein is known to couple a large number of G protein-coupled receptors to PLC-ß and can lead to production of the secondary messengers diacylglycerol and inositol phosphates. Targeted deletion of Ga 15, a mouse orthologue of human Ga 16, causes reduced C5a receptor signaling. Ga 16serves as a marker, in addition to CD34, for hematopoietic progenitor cells.
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