Product Pathways - Tyrosine Kinase/ Adaptors
HER4/ErbB4 Kinase #7724
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Description
Purified recombinant human HER4/ErbB4 kinase (Arg676-Val1308), supplied as a GST fusion protein.
Source / Purification
The GST-kinase fusion protein was produced using a baculovirus expression system with a construct expressing human HER4/ErbB4 (Arg676-Val1308) (GenBank accession No. NM_005235) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-HER4/ErbB4 fusion protein was analyzed using SDS/PAGE followed by anti-HER4/ErbB4 Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. HER4/ErbB4 kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20,000, Substrate: PolyEY, 5 µg/50 µl and 50 ng/50 µl Recombinant HER4/ErbB4.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of HER4/ErbB4 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1315) by HER4/ErbB4 kinase. In a 50 µl reaction, increasing amounts of HER4/ErbB4 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Quality Control
The theoretical molecular weight of the GST-HER4/ErbB4 fusion protein is 102.4 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. HER4/ErbB4 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure HER4/ErbB4 activity using HTScan™ HER4/ErbB4 Kinase Assay Kit #7725 [Fig.3].
Background
The HER/ErbB receptor tyrosine kinase family has been implicated in normal development, cardiac function and cancer (1,2). HER4/ErbB4, like other family members, has four ectodomains, a single transmembrane domain and a cytoplasmic tail containing the active tyrosine kinase domain (3). By binding to neuregulins and/or EGF family ligands, ErbB4 forms either a homodimer or heterodimer with other ErbB family members, which results in receptor activation and signaling (3). ErbB4 is ubiquitously expressed with the highest expression occurring in brain and heart. The expression of ErbB4 in breast cancer, pediatric brain cancer and other types of carcinomas has been reported suggesting that ErbB4 expression is involved in both normal tissue development and carcinogenesis (3).
- Holbro, T. and Hynes, N.E. (2004) Annu. Rev. Pharmacol. Toxicol. 44, 195-217.
- Roskoski Jr, R. (2004) Biochem. Biophys. Res. Commun. 319, 1-11.
- Carpenter, G. (2003) Exp. Cell Res. 284, 66-77.
Application References
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