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Ga16 抗体

ABIN110590 产品详细信息, 供应商: Log in to see
抗原
适用

宿主

克隆类型
多克隆
应用范围
Immunoprecipitation (IP), Western Blotting (WB)
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'独立验证'标志
抗原 Ga16
Lot Number 82100
Method validated Western Blotting
Positive Control QGP1 and PT45 lysates
Negative Control MIA PaCa-2 cells lysate; PT45 shRNA knock-down cell lysate
Primary Antibody ABIN110590
Secondary Antibody goat anti-rabbit IgG HRP antibody (Sigma-Aldrich, A0545)
实验流程
  • 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.
    Experimental 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.
    Validation Images
    Western Blotting validation image for anti-Ga16 antibody (ABIN110590) Immunoblot in TBST using milk 5% as blocking agent; see protocol for details. A. Lysa...
    背景 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.
    限制 仅限研究用
    状态 Lyophilized
    储存条件 4 °C