Western blot analysis of extracts from cells expressing various activated tyrosine kinase proteins, using Phospho-FLT3 (Tyr589/591) (30D4) Rabbit mAb (upper) or Phospho-Tyrosine mAb (P-Tyr-100) #9411 (lower). Phospho-FLT3 (Tyr589/591) (30D4) Rabbit mAb specifically recognizes the activated FLT3 but not non-phosphorylated FLT3 protein.
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-FLT3 (Tyr589/591) (30D4) Rabbit mAb detects endogenous levels of FLT3 protein only when phosphorylated at tyrosine 589/591. The antibody does not cross-react with other phosphorylated tyrosine kinases. (US Patent No. 7,183,385 and foreign equivalents assigned to Cell Signaling Technology, Inc.)
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to the sequences surrounding Tyr589/591 of human FLT3.
FMS-related tyrosine kinase 3 (FLT3, also called Flk2), is a member of the type III receptor tyrosine kinase family, which includes c-Kit, PDGFR and M-CSF receptors. FLT3 is expressed on early hematopoietic progenitor cells and supports growth and differentiation within the hematopoietic system (1,2). FLT3 is activated after binding with its ligand FL, which results in a cascade of tyrosine autophosphorylation and tyrosine phosphorylation of downstream targets (3). The p85 subunit of PI3 kinase, SHP2, GRB2 and Shc are associated with FLT3 after FL stimulation (4-6). Tyr589/591 is located in the juxtamembrane region of FLT3 and may play an important role in regulation of FLT3 tyrosine kinase activity. Somatic mutations of FLT3 consisting of internal tandem duplications (ITDs) occur in 20% of patients with acute myeloid leukemia (7).
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