Western blot analysis of extracts from 293 cells overexpressing human FGFR-1, untreated or calf intestinal phosphatase (CIP)-treated, using Phospho-FGF Receptor (Tyr653/654) Antibody (upper) or FGF Receptor 1 Antibody #3472 (lower). Overexpression of human FGFR-1 protein in 293 cells results in constitutive activation of the receptors (courtesy of Dr. Pamela Maher, personal communication). CIP treatment abolishes the reactivity of this antibody to FGFR-1.Learn more about how we get our images.
Western blot analysis of extracts from A-204 cells treated with or without bFGF, using Phospho-FGF Receptor (Tyr653/654) Antibody (upper) and FGF Receptor 1 (D8E4) XP® Rabbit mAb (lower).Learn more about how we get our images.
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
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.
Phospho-FGF Receptor (Tyr653/654) Antibody detects endogenous levels of FGF receptors only when phosphorylated at tyrosines 653/654. This antibody cross-reacts with activated PDGF receptor and insulin/IGF-I receptors.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr653/654 of human FGFR-1 (the corresponding sequence is the same in FGFR-2, -3 and -4). Antibodies are purified by protein A and peptide affinity chromatography.
Fibroblast growth factors (FGFs) produce mitogenic and angiogenic effects in target cells by signaling through cell surface receptor tyrosine kinases. There are four members of the FGF receptor family: FGFR1 (flg), FGFR2 (bek, KGFR), FGFR3, and FGFR4. Each receptor contains an extracellular ligand binding domain, a transmembrane domain, and a cytoplasmic kinase domain (1). Following ligand binding and dimerization, the receptors are phosphorylated at specific tyrosine residues (2). Seven tyrosine residues in the cytoplasmic tail of FGFR1 can be phosphorylated: Tyr463, 583, 585, 653, 654, 730, and 766. Tyr653 and Tyr654 are important for catalytic activity of activated FGFR and are essential for signaling (3). The other phosphorylated tyrosine residues may provide docking sites for downstream signaling components such as Crk and PLCγ (4,5).
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