ErbB2, phosphorylated, Tyr1248 (c-erbB2, HER2, neu Oncoprotein) (FITC)

Size

£1,023.00

SKU:: E3451-25-FITC
Application:: WB
Purity:: Purified by immunoaffinity chromatography.
Storage Conditions:: -20°C
Supplier:: United States Biological
Host:: Rabbit
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
Format:: Supplied as a liquid in PBS, pH 7.2. No preservative added. Labeled with Fluorescein isothiocyanate (FITC).
Extra Details:: Applications: Suitable for use in Western Blot. Other applications not tested. Recommended Dilution: Western Blot: 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: Store product at 4°C if to be used immediately within two weeks. For long-term storage, aliquot to avoid repeated freezing and thawing and store at -20°C. Aliquots are stable at -20°C for 12 months after receipt. Dilute required amount only prior to immediate use. Further dilutions can be made in assay buffer. Caution: FITC conjugates are sensitive to light. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Note: Applications are based on unconjugated antibody.

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