Ga16 antibody
Reactivity: Human
IP, WB
Host: Rabbit
Polyclonal
unconjugated
1 image
(1 validation)-
- Target
- Ga16
- Reactivity
- Human
- Host
- Rabbit
- Clonality
- Polyclonal
- Application
- Immunoprecipitation (IP), Western Blotting (WB)
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- Restrictions
- For Research Use only
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- by
- Laboratorio Universitario di Ricerca Medica, Univsersità degli Studi di Verona
- No.
- #100205
- Date
- 11/27/2016
- Antigen
- 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
- Notes
- Primary Antibody
- ABIN110590
- Secondary Antibody
- 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.
- 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 #100205 (Western Blotting)!['Independent Validation' Badge]( "Successfully validated")
!['Independent Validation' Badge]( "Successfully validated")
Validation Images![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.]()
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.
Full Methods -
- Format
- Lyophilized
- Storage
- 4 °C
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- Target
- Ga16
- Background
- 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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