Anti-EGF-Receptor (Epidermal Growth Factor Recptor, erbB, EGFR) (nonPhospho-Y1173) (Glycerol and azi

Applications:, Suitable for use in Immunoprecipitation and Western Blot. Other applications have not been tested. Recommended Dilution: Immunoprecipitation: 1-10ug Reported to immunoprecipitate the EGF receptor (unphosphorylated at Y1173) from 10e6 vanadate treated A431 cells. , Immunoblot Analysis: 0.5-2ug/ml detects the EGF receptor (unphosphorylated at Y1173) in RIPA lysates from unstimulated HFF cells. Lysates from EGF stimulated and unstimulated HFF cells, 10ug per lane, were resolved by electrophoresis and transferred to nitro-cellulose. Probed with EGFR visualized using a rabbit anti-sheep secondary antibody conjugated to HRP. Probed with E3375-16X (non-phospho-Y1173) (1ug/ml) and visualized using a goat anti-mouse secondary antibody conjugated to HRP. A chemiluminescence detection system was used. (EGF-R 175 kD). , Optimal dilutions to be 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 20 minutes at RT with constant agitation. 3. Incubate the nitrocellulose with 0.5-2ug/ml of anti-EGF-R (non-phospho-Y1173), 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-mouse HRP conjugated IgG, 1:2000 dilution was used) in PBS-MLK for 1.5 hours at RT with agitation. 6. Wash the nitrocellulose with water twice. 7. Wash the nitrocellulose in PBS-0.05% Tween 20 for 3-5 minutes. 8. Rinse the nitrocellulose in 4-5 changes of water. 9. Use detection method of choice (enhanced chemiluminescence was used). Storage and Stability: May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.