Western blot analysis of extracts from overnight serum-starved H526 cells, untreated (-) or treated with hSCF #8925 (100 ng/ml, 5 min; +), using Phospho-c-Kit (Tyr568/570) Antibody (upper) and c-Kit (D13A2) XP® Rabbit mAb #3074 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. 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-c-Kit (Tyr568/570) Antibody recognizes endogenous levels of c-Kit protein only when phosphorylated at Tyr568/570. This antibody also recognizes a hSCF-induced, non-specific band at 95 kDa from a protein of unknown identity.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr568/570 of human c-Kit protein. Antibodies are purified by protein A and peptide affinity chromatography.
c-Kit is a member of the subfamily of receptor tyrosine kinases that includes PDGF, CSF-1, and FLT3/flk-2 receptors (1,2). It plays a critical role in activation and growth in a number of cell types including hematopoietic stem cells, mast cells, melanocytes, and germ cells (3). Upon binding with its stem cell factor (SCF) ligand, c-Kit undergoes dimerization/oligomerization and autophosphorylation. Activation of c-Kit results in the recruitment and tyrosine phosphorylation of downstream SH2-containing signaling components including PLCγ, the p85 subunit of PI3 kinase, SHP2, and CrkL (4). Molecular lesions that impair the kinase activity of c-Kit are associated with a variety of developmental disorders (5), and mutations that constitutively activate c-Kit can lead to pathogenesis of mastocytosis and gastrointestinal stromal tumors (6). Tyr719 is located in the kinase insert region of the catalytic domain. c-Kit phosphorylated at Tyr719 binds to the p85 subunit of PI3 kinase in vitro and in vivo (7).
Tyr568 and Tyr570 are located in the juxtamembrane region of c-Kit. The phosphorylation of c-Kit at these sites is ligand induced and provides a docking site for recruitment of several adaptors/kinases, including Src, Cbl, SHP-1, APS, LNK, and SOSC6. Depending on which adaptor is bound, the outcome can lead to either cell survival and proliferation or c-Kit ubiquitination and degradation (8-13).
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