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

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).

Anti-MDM2 (Ab-1) Mouse mAb (IF2) MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: Anti-Ubiquitin Protein Ligase, Anti-p53 Binding Protein, Anti-Murine Double Minute Chromosome-2

OP46
Purchase on Sigma-Aldrich

Przegląd

Replacement Information

Tabela kluczowych gatunków

Species ReactivityHostAntibody Type
HMMonoclonal Antibody

Products

Numer katalogowyOpakowanie Ilość/opak.
OP46-100UG Ampulka plastikowa 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-Ubiquitin Protein Ligase, Anti-p53 Binding Protein, Anti-Murine Double Minute Chromosome-2
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
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Storage and Shipping Information
Ship Code Blue Ice Only
Toxicity Standard Handling
Storage +2°C to +8°C
Do not freeze Yes
Packaging Information
Transport Information
Supplemental Information
Specifications
Global Trade Item Number
Numer katalogowy GTIN
OP46-100UG 04055977227215

Documentation

Anti-MDM2 (Ab-1) Mouse mAb (IF2) Certificates of Analysis

TitleLot Number
OP46

References

Przegląd literatury
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. 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.

Brochure

Title
Caspases and other Apoptosis Related Tools Brochure

Citations

Tytuł
  • Hazel E. Warburton, et al. (2005) p53 regulation and function in renal cell carcinoma. Cancer Research 65, 6498-6503.
    		•
    Data Sheet

    Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

    Revision10-September-2008 JSW
    SynonymsAnti-Ubiquitin Protein Ligase, Anti-p53 Binding Protein, Anti-Murine Double Minute Chromosome-2
    ApplicationFrozen 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)
    DescriptionPurified mouse monoclonal antibody (see application references). Recognizes the ~90 kDa (apparent MW) MDM2 protein. Also recognizes isoforms at ~57 and ~74/76 kDa.
    BackgroundIncreased expression of proto-oncogenes due to gene amplification can lead to cellular transformation in the absence of other mutagenic events. Recently, a novel gene termed MDM2, first identified in mouse as a small acentromeric extrachromosomal element, was shown to become oncogenic upon amplification and tumorigenic when overexpressed in NIH3T3 and Rat2 cells. The human homolog of MDM2 gene has been isolated and mapped to chromosome 12q13-q14, a region that is found to be amplified in human sarcomas. Sequence analysis of the murine, rat, and human MDM2 genes suggests that MDM2 may be involved in transcriptional regulation. MDM2 contains two putative metal-binding motifs, one of which shares similarity to known zinc finger transcriptional activators. MDM2 also contains a putative nuclear localization signal and an acidic domain of the type found in transcriptional activators. A functional role for MDM2 has recently been identified on the basis of experiments which demonstrate that MDM2 forms a stable complex with p53 in vivo. Furthermore, over-expression of MDM2 inhibits the ability of wild type p53 to stimulate expression of target genes, and this inhibition appears to result from MDM2 binding to the acidic activation domain of p53, thereby preventing p53 from directly contacting the transcriptional machinery. Interestingly, wild type p53 appears to positively regulate expression of the MDM2 gene. MDM2 amplification has been observed in sarcomas, and direct analysis of the MDM2 and p53 genes in sarcomas indicates that one or the other but not both of these genes is mutated in 70% of the tumors. Thus, it appears that genetic alterations in either p53 or MDM2 represent alternative mechanisms for inactivating the same growth suppressive pathway. Regulation of MDM2 expression by p53 also represents a potential feed back control of p53 function.
    HostMouse
    Immunogen speciesHuman
    Immunogenhuman MDM2
    Epitopewithin amino acids 26-169 of human MDM2
    CloneIF2
    IsotypeIgG2b
    Specieshuman, not mouse
    Positive controlOSA-CL cells
    FormLiquid
    FormulationIn 50 mM sodium phosphate buffer, 0.2% gelatin.
    Concentration Label Please refer to vial label for lot-specific concentration
    Preservative≤0.1% sodium azide (100 μg only)
    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.
    Storage +2°C to +8°C
    Do Not Freeze Yes
    Toxicity Standard Handling
    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.
    Citation
  • Hazel E. Warburton, et al. (2005) p53 regulation and function in renal cell carcinoma. Cancer Research 65, 6498-6503.
    		•
    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.