Ga16 Antikörper

Produktnummer ABIN110590

Dieses unabhängig validierte Kaninchen Polyklonal-Antikörper erkennt spezifisch in IP und WB. Er zeigt eine Reaktivität gegenüber Human.

Kurzübersicht für Ga16 Antikörper (ABIN110590)

Target: Ga16

Reaktivität: Human

Wirt: Kaninchen

Klonalität: Polyklonal

Applikation: Immunoprecipitation (IP), Western Blotting (WB)

Produktdetails

Anwendungsinformationen

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (WB)

Validierung #100205 (Western Blotting)

by: Laboratorio Universitario di Ricerca Medica, Univsersità degli Studi di Verona

No.: #100205

Datum: 27.11.2016

Antigen: Ga16

Chargennummer: 82100

Validierte Anwendung: Western Blotting

Positivkontrolle: QGP1 and PT45 lysates

Negativkontrolle: MIA PaCa-2 cells lysate; PT45 shRNA knock-down cell lysate

Validierungsbilder

Immunoblot in TBST using milk 5% as blocking agent; see protocol for details. A. Lysate from pancreatic cancer cell lines was loaded as indicated. B. Lysate from PT45 cells, untreated in lane 1 and treated with shRNA specific for GNA15 in lane 2.

Protokoll

Primärantikörper: ABIN110590

Sekundärantikörper: 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.

Anmerkungen: 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.

Handhabung

Format: Lyophilized

Lagerung: 4 °C

Antigendetails

Target: Ga16

Hintergrund: 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.