This gene encodes a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases.
This protein has no ligand binding domain of its own and therefore cannot bind growth factors.
However, it does bind tightly to other ligand-bound EGF receptor family members to form a heterodimer, stabilizing ligand binding and enhancing kinase-mediated activation of downstream signalling pathways, such as those involving mitogen-activated protein kinase and phosphatidylinositol-3 kinase.
Allelic variations at amino acid positions 654 and 655 of isoform a (positions 624 and 625 of isoform b) have been reported, with the most common allele, Ile654/Ile655, shown here.
Amplification and/or overexpression of this gene has been reported in numerous cancers, including breast and ovarian tumors.
Alternative splicing results in several additional transcript variants, some encoding different isoforms and others that have not been fully characterized.
Applications:Suitable for use in Western Blot.
Other applications not tested.
Recommended Dilution:Western Blot: 1:2000 -1:8000 dilutions detects activated Erb B2/HER-2 in RIPA lysates from heregulin stimulated MCF-7 breast carcinoma cells.
Lysates from non-stimulated and heregulin-stimulated MCF-7 cells were resolved by electrophoresis, transferred to nitrocellulose and probed with E3451-25 (1:2000 dilution).
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system
Optimal dilution determined by the researcher.
Immunoblot Protocol 1.
Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a cell lysate sample (cell lysis buffer: 50mM Tris-HCl, pH7.4; 1% NP-40; 0.25% sodium deoxycholate; 150mM NaCl; 1mM EGTA; 1mM PMSF; 1ug/ml each aprotinin, leupeptin, pepstatin; 1mM Na3VO4; 1mM NaF) and transfer the proteins to nitrocellulose.
Wash the blotted nitrocellulose twice with water.
2.
Block the blotted nitrocellulose in freshly prepared PBS containing 3% nonfat dry milk (PBS-MLK) for 30 minutes at RT with constant agitation.
3.
Incubate the nitrocellulose with a 1:2000-1:8000 dilution of E3451-25 diluted in freshly prepared PBS-MLK overnight with agitation at 4°C.
4.
Wash the nitrocellulose twice with water.
5.
Incubate the nitrocellulose in the secondary reagent of choice (a goat anti-rabbit HRP conjugated IgG, 1:5000 dilution was used) in PBS-MLK for 1.5 hours at room temperature with agitation.
6.
Wash the nitrocellulose with water twice.
7.
Wash the nitrocellulose in PBS-0.05% Tween 20 for 3-15 minutes.
8.
Rinse the nitrocellulose in water for 30-60min.
9.
Use detection method of choice (enhanced chemiluminescence was used).
Storage and Stability:May be stored at 4°C for short-term only.
Aliquot to avoid repeated freezing and thawing.
Store at -20°C.
Aliquots are stable for 12 months after receipt.
For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
仕様
Size：100ul
Host：rabbit
Source Antibody：human
Grade：Affinity Purified
Purity：Purified by immunoaffinity chromatography.
Form：Supplied as a liquid in 0.2M Tris-glycine, pH 7.4, 0.15M sodium chloride, 0.05% sodium azide.
Specificity：Reecognizes the C-terminus of human erbB-2/HER-2 phosphorylated at Tyr1248.
Isotype：IgG
Calc Applications Abbrev：WB
Calc Crossreactivity：Hu
Immunogen：Synthetic phosphopeptide, residues 1243-1255, corresponding to a major auto-phosphorylation (Tyr1248) site of human erbB-2/HER-2 (C-AENPEpYLGLDVPV). Species sequence homology (100%): mouse, rat and monkey
