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OP46 Anti-MDM2 (Ab-1) Mouse mAb (IF2)

Overview

Key Specifications Table

Species ReactivityHostAntibody Type
HMMonoclonal Antibody

Products

Catalog NumberPackaging Qty/Pack
OP46-100UG Plastic ampoule 100 μg
Description
OverviewRecognizes the ~90 kDa (apparent MW) MDM2 protein. Also recognizes isoforms of ~57 kDa and ~74/76 kDa by immunoblotting.
Catalogue NumberOP46
Brand Family Calbiochem®
SynonymsAnti-Murine Double Minute Chromosome-2, Anti-Ubiquitin Protein Ligase, Anti-p53 Binding Protein
References
ReferencesGorgoulis, 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. Nature 362, 857.
Momand, J., et al. 1992. Cell 69, 1237.
Oliner, J.D., et al. 1992. Nature 358, 80.
Fakharzadeh, S.S., et al. 1991. EMBO J. 10, 1565.
Product Information
FormLiquid
FormulationIn 50 mM sodium phosphate buffer, 0.2% gelatin.
Positive controlOSA-CL cells
Preservative≤0.1% sodium azide (100 μg only)
Quality LevelMQ100
Applications
Application ReferencesOriginal Clone, Frozen Sections Leach, F. S., et al. 1993. Cancer Res. 53, 2231. Paraffin Sections Marchetti, A., et al. 1995. J. Pathol. 175, 31.
Key Applications Frozen Sections
Immunoblotting (Western Blotting)
Immunofluorescence
Immunoprecipitation
Paraffin Sections
Application NotesFrozen 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)
Application CommentsAlthough 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.
Biological Information
Immunogenhuman MDM2
ImmunogenHuman
Epitopewithin amino acids 26-169 of human MDM2
CloneIF2
HostMouse
IsotypeIgG2b
Species Reactivity
  • Human
Antibody TypeMonoclonal Antibody
Concentration Label Please refer to vial label for lot-specific concentration
Storage and Shipping Information
Ship Code Blue Ice Only
Toxicity Standard Handling
Storage +2°C to +8°C
Do not freeze Yes
Global Trade Item Number
Catalog Number GTIN
OP46-100UG 04055977227215