Western blot analysis of extracts from NCI-H1299 cells, untreated or Gas6-treated (100 ng/ml for 10 minutes), using Phospho-Axl (Tyr702) (D12B2) Rabbit mAb (upper) or Axl (C44G1) Rabbit mAb #4566 (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 BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: 10
Phospho-Axl (Tyr702) (D12B2) Rabbit mAb detects endogenous levels of Axl only when phosphorylated at Tyr702. This antibody may also cross-react with other overexpressed, related tyrosine-phosphorylated tyrosine kinases, such as EGFR.
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr702 of human Axl protein.
Axl, Sky, and Mer are three members of a receptor tyrosine kinase (RTK) family that share a conserved intracellular tyrosine kinase domain and an extracellular domain similar to those seen in cell adhesion molecules. These RTKs bind the vitamin K-dependent protein growth-arrest-specific 6 (Gas6), which is structurally related to the protein S anticoagulation factor (1). Upon binding to its receptor, Gas6 activates phosphatidylinositol 3-kinase (PI3K) and its downstream targets Akt and S6K, as well as NF-κB (2,3). A large body of evidence supports a role for Gas6/Axl signaling in cell growth and survival in normal and cancer cells (4).
Phosphorylation of Axl Receptor on Tyr702 was identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's LC-MS/MS platform for phosphorylation site discovery (5). Phosphorylation of Axl on Tyr702 was also reported in select carcinoma cell lines (6).
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