Western blot analysis of extracts from MDA-MB-231 (HER3-) and MCF7 (HER3+) cells using HER3/ErbB3 (D22C5) XP® Rabbit mAb (HRP Conjugate).
This Cell Signaling Technology antibody is conjugated to the carbohydrate groups of horseradish peroxidase (HRP) via its amine groups. The HRP conjugated antibody is expected to exhibit the same species cross-reactivity as the unconjugated HER3/ErbB3 (D22C5) XP® Rabbit mAb #12708.
Supplied in 136 mM NaCl, 2.6 mM KCl, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
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 product webpage for recommended antibody dilution.
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 June 2020
Protocol Id: 11
HER3/ErbB3 (D22C5) XP® Rabbit mAb (HRP Conjugate) recognizes endogenous levels of total HER3/ErbB3 protein. This antibody does not cross-react with other HER family proteins.
Monoclonal antibody is produced by immunizing animals with recombinant protein corresponding to the carboxy terminus of human ErbB3 protein.
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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