Western blot analysis of extracts from COS cells overexpressing human FGF receptor-1, untreated or calf intestine phosphatase (CIP)-treated, using Phospho-FGF Receptor (Tyr653/654) (55H2) Mouse mAb. Overexpression of human FGF receptor-1 results in constitutive activation of the receptors (courtesy of Dr. Pamela Maher, Scripps Research Institute, California, personal communication). CIP treatment abolishes the reactivity of this antibody to FGF receptor-1.
Western blot analysis of extracts from A-204 cells treated with or without bFGF, using Phospho-FGF Receptor (Tyr653/654) (55H2) Mouse mAb (upper) and FGF Receptor 1 (D8E4) XP® Rabbit mAb (lower).
|MW (kDa)||120, 145|
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 nonfat dry milk, 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 June 2016
Protocol Id: 19
Phospho-FGF Receptor (Tyr653/654) (55H2) Mouse mAb detects endogenous levels of FGF receptors only when phosphorylated at tyrosines 653/654. This antibody detects phosphorylated FGF Receptors 2 and 4 when expressed exogenously. Based on sequence comparisons, reactivity with FGF Receptor 3 is possible but has not been experimentally confirmed. The antibody also cross-reacts slightly with activated PDGF and insulin/IGF-I receptors.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 Tyr653/654 of human FGF receptor-1. The corresponding sequence is identical in FGF receptor-2, -3 and -4.
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).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.