Western blot analysis of cell extracts from Baf3/FLT3 cells, untreated or treated with FLT3 ligand (FL), using Phospho-FLT3 (Tyr969) (C24D9) Rabbit mAb (upper) or FLT3 (8F2) Rabbit mAb Antibody #3462 (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-FLT3 (Tyr969) (C24D9) Rabbit mAb detects endogenous levels of FLT3 only when phosphorylated at Tyr969. The antibody may cross-react with other tyrosine-phosphorylated proteins. (US Patent No. 7,183,385 and foreign equivalents assigned to Cell Signaling Technology, Inc.)Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to the sequence surrounding Tyr969 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).
Phospho-FLT3 (Tyr969) (C24D9) Rabbit mAb is directed against a previously unpublished FLT3 tyrosine phosphorylation site at Tyr969 that was identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's MS/MS platform for phosphorylation site discovery. Phosphorylation of FLT3 at Tyr969 was observed in various leukemia, lymphoma and carcinoma cell lines and in tumors.
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.