Western blot analysis of extracts from various cell lines using Ras (G12V Mutant Specific) (D2H12) Rabbit mAb (upper), Ras (D2C1) Rabbit mAb #8955 (middle), and β-Actin (D6A8) #8457 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
Ras (G12V Mutant Specific) (D2H12) Rabbit mAb recognizes endogenous levels of Ras G12V mutant protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to G12V mutant sequence of human Ras protein.
The 21 kDa guanine-nucleotide binding proteins (K-Ras, H-Ras, and N-Ras) cycle between active (GTP-bound) and inactive (GDP-bound) forms (1). Receptor tyrosine kinases and G protein-coupled receptors activate Ras, which then stimulates the Raf-MEK-MAPK pathway (2-4). GTPase-activating proteins (GAP) normally facilitate the inactivation of Ras. However, research studies have shown that in 30% of human tumors, point mutations in Ras prevent the GAP-mediated inhibition of this pathway (5). The most common oncogenic Ras mutation found in tumors is Gly12 to Asp12 (G12D), which prevents Ras inactivation, possibly by increasing the overall rigidity of the protein (5,6).
Additional oncogenic mutations of Ras have been observed at varying frequencies at codons 12, 13, and 61. The Gly12 to Val12 (G12V) mutation has been detected in a number of different cancers, including colorectal and thyroid cancer (7,8).
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