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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
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.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
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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