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OP46

Sigma-Aldrich

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

liquid, clone IF2, Calbiochem®

Synonym(s):

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

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About This Item

UNSPSC Code:
12352203
NACRES:
NA.41

biological source

mouse

Quality Level

antibody form

purified antibody

antibody product type

primary antibodies

clone

IF2, monoclonal

form

liquid

contains

≤0.1% sodium azide as preservative (100 μg only)

species reactivity

human

should not react with

mouse

manufacturer/tradename

Calbiochem®

storage condition

do not freeze

isotype

IgG2b

shipped in

wet ice

storage temp.

2-8°C

target post-translational modification

unmodified

Gene Information

human ... MDM2(4193)
mouse ... Mdm2(17246)

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)

Packaging

Please refer to vial label for lot-specific concentration.

Warning

Toxicity: Standard Handling (A)

Physical form

In 50 mM sodium phosphate buffer, 0.2% gelatin.

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.
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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.

Legal Information

CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany
TWEEN is a registered trademark of Croda International PLC

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Storage Class Code

10 - Combustible liquids

WGK

nwg

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable


Certificates of Analysis (COA)

Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.

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Oncogenic MYC activation promotes proliferation in Burkitt lymphoma, but also induces cell-cycle arrest and apoptosis mediated by p53, a tumor suppressor that is mutated in 40% of Burkitt lymphoma cases. To identify molecular dependencies in Burkitt lymphoma, we performed RNAi-based

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