Description General description Purified mouse monoclonal antibody (see application references). Recognizes the ~90 kDa (apparent MW) MDM2 protein. Also recognizes isoforms at ~57 and ~74/76 kDa. Recognizes the ~90 kDa (apparent MW) MDM2 protein. Also recognizes isoforms of ~57 kDa and ~74/76 kDa by immunoblotting. This Anti-MDM2 (Ab-1) Mouse mAb (IF2) is validated for use in Frozen Sections Immunoblotting Immunofluorescence Immunoprecipitation Paraffin Sections for the detection of MDM2 (Ab-1). Immunogen Epitope: within amino acids 26-169 of human MDM2 Human human MDM2 Application Frozen Sections (1-5 µg/ml, see application references) Immunoblotting (0.5-2 µg/ml, chemiluminescence) Immunofluorescence (1-5 µg/ml) Immunoprecipitation (1 µg/sample) Paraffin Sections (1-5 µg/ml, heat pre-treatment required, see application references) Physical form In 50 mM sodium phosphate buffer, 0.2% gelatin. Legal Information TWEEN is a registered trademark of Croda International PLC Packaging 100 μg in Plastic ampoule Please refer to vial label for lot-specific concentration. Warning Toxicity: Standard Handling (A) Analysis Note Positive Control OSA-CL cells Other Notes Although the amino acid sequence of MDM2 predicts a protein with a molecular mass of approximately 54 kDa, MDM2 protein migrates on SDS/PAGE with an apparent mobility of 90 kDa. Immunoblotting Protocol MDM2 (Ab-1) can be used to detect MDM2 by Western blot of proteins previously separated by SDS/PAGE and electrophoretically transferred onto nitrocellulose membranes. The proteins are reacted with the monoclonal antibody and visualized using an HRP conjugated goat anti-mouse antibody with chemiluminescent detection. Materials Equipment: • Electrophoresis apparatus • Electroblotting apparatus • Rocker platform Solutions and Reagents • Anti-MDM2 (Ab-1) Mouse mAb (IF2) Cat. No. OP46 or OP46T • HRP conjugated goat anti-mouse IgG heavy and light chains (e.g. Cat. No. 401215) • Chemiluminescence detection system • ELB Buffer (include a cocktail of proetease inhibitors, such as 0.5 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM EDTA and 0.2 mM PMSF): 50 mM Hepes pH 7.0, 250 mM NaCl, 0.5 mM EDTA, 0.1% Nonidet P-40 Alternative • SDS-PAGE (7% acrylamide) • Phosphate buffered saline (PBS) pH 7.4; 1 Liter: 0.2 g KCl, 0.2 g KH2PO4, 8 g NaCl, 1.15 g Na2HPO4 • PBS/0.1% Tween®-20 detergent (PBST) • 3% Non-fat Dry Milk in PBST Procedure 1. Lyse cells in ELB Buffer. (Alternatively, cells can be lysed in RIPA Buffer or directly into 1x Laemmli Sample Buffer). 2. Electrophorese 50-100 µg lysate using a 7% acrylamide gel. 3. Transfer the protein samples from the polyacrylamide gel onto a nitrocellulose membrane using an electroblotting apparatus. 4. Block the membrane for 1 h in PBST containing 3% non-fat dry milk at room temperature with rocking. Use about 1 ml per cm2 of membrane. 5. Incubate the membrane with 1 µg/ml Anti-MDM2 (Ab-1) Mouse mAb (IF2) in 3% non-fat dry milk/ PBST for 1 h at room temperature with rocking. 6. Wash the membrane 3 times, 15 min each, in PBST at room temperature with rocking. 7. Incubate the membrane with HRP conjugated goat anti-mouse IgG heavy and light chain antibody, diluted according to the supplier’s instructions, in 3% non-fat dry milk/ PBST at room temperature for 1 h. 8. Wash the membrane 4 times, 15 min each, in PBST at room temperature with rocking. 9. Develop the membrane using chemiluminescent detection reagents according to manufacturer instructions. 10. Expose the membrane to film for ten minutes. Adjust subsequent exposure times as needed. Gorgoulis, V.G., et al. 1996. J. Pathol.180, 129. Marchetti, A., et al. 1995. J. Pathol.175, 31. Barak, Y., et al. 1993. EMBO J.12, 461. Ladanyi, M., et al. 1993. Cancer Res.53, 16. Leach, F.S., et al. 1993. Cancer Res.53, 2231. Oliner, J.D., et al. 1993. Nature362, 857. Momand, J., et al. 1992. Cell69, 1237. Oliner, J.D., et al. 1992. Nature358, 80. Fakharzadeh, S.S., et al. 1991. EMBO J. 10, 1565.