Cross-reactivity testing of Phospho-Insulin Receptor (Tyr1361) (84B2) Rabbit mAb with activated IGF-I receptors. Various amounts of activated recombinant IGF-I receptor cytoplasmic domain proteins were applied for Western blot analysis, using Phospho-Insulin Receptor (Tyr1361) (84B2) Rabbit mAb (upper) and Phospho-IGF-I Receptor (Tyr1315/1316) (19H7) Rabbit mAb #3024 (lower) respectively. The result showed that Phospho-Insulin Receptor (Tyr1361) (84B2) Rabbit mAb barely cross-reacts with activated IGF-I receptors.
Western blot analysis of CHO cells overexpressing human insulin receptors untreated or treated with insulin (100 nM for 5 min), using Phospho-Insulin Receptor β (Tyr1361) (84B2) Rabbit mAb (upper) and Insulin Receptor β (4B8) Rabbit mAb #3025(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-Insulin Receptor β (Tyr1361) (84B2) Rabbit mAb detects transfected levels of insulin receptor β only when phosphorylated at Tyr1361. It slightly cross-reacts with activated IGF-I receptor, but does not cross-react with other activated tyrosine kinases.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr1361 of human insulin receptor β.
Type I insulin-like growth factor receptor (IGF-IR) is a transmembrane receptor tyrosine kinase that is widely expressed in many cell lines and cell types within fetal and postnatal tissues (1-3). Receptor autophosphorylation follows binding of the IGF-I and IGF-II ligands. Three tyrosine residues within the kinase domain (Tyr1131, Tyr1135, and Tyr1136) are the earliest major autophosphorylation sites (4). Phosphorylation of these three tyrosine residues is necessary for kinase activation (5,6). Insulin receptors (IRs) share significant structural and functional similarity with IGF-I receptors, including the presence of an equivalent tyrosine cluster (Tyr1146/1150/1151) within the kinase domain activation loop. Tyrosine autophosphorylation of IRs is one of the earliest cellular responses to insulin stimulation (7). Autophosphorylation begins with phosphorylation at Tyr1146 and either Tyr1150 or Tyr1151, while full kinase activation requires triple tyrosine phosphorylation (8).
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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.