Western blot analysis of MCF-7 and T47D cell lysates untreated or stimulated with Neuregulin (100 ng/ml) for 15 min, using Phospho-HER3/ErbB3 (Tyr1222) (50C2) Rabbit mAb (upper) and HER3/ErbB3 (1B2) Rabbit mAb #4754 (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-HER3/ErbB3 (Tyr1222) (50C2) Rabbit mAb detects endogenous HER3/ErbB3 proteins only when phosphorylated at tyrosine 1222. The antibody does not cross-react with other tyrosine phosphorylated receptor tyrosine kinases.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr1222 of human HER3/ErbB3.
HER3/ErbB3 is a member of the ErbB receptor protein tyrosine kinase family, but it lacks tyrosine kinase activity. Tyrosine phosphorylation of ErbB3 depends on its association with other ErbB tyrosine kinases. Upon ligand binding, heterodimers form between ErbB3 and other ErbB proteins, and ErbB3 is phosphorylated on tyrosine residues by the activated ErbB kinase (1,2). There are at least 9 potential tyrosine phosphorylation sites in the carboxy-terminal tail of ErbB3. These sites serve as consensus binding sites for signal transducing proteins, including Src family members, Grb2, and the p85 subunit of PI3 kinase, which mediate ErbB downstream signaling (3). Both Tyr1222 and Tyr1289 of ErbB3 reside within a YXXM motif and participate in signaling to PI3K (4).
Investigators have found that ErbB3 is highly expressed in many cancer cells (5) and activation of the ErbB3/PI3K pathway is correlated with malignant phenotypes of adenocarcinomas (6). Research studies have demonstrated that in tumor development, ErbB3 may function as an oncogenic unit together with other ErbB members (e.g. ErbB2 requires ErbB3 to drive breast tumor cell proliferation) (7). Thus, investigators view inhibiting interaction between ErbB3 and ErbB tyrosine kinases as a novel strategy for anti-tumor therapy.
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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.