CDC25A antibody

Catalog No. ABIN487337

The Mouse Monoclonal anti-CDC25A antibody has been validated for WB, IHC (p) and IP. It is suitable to detect CDC25A in samples from Human, Mouse and Rat.

Quick Overview for CDC25A antibody (ABIN487337)

Target: CDC25A (Cell Division Cycle 25 Homolog A (S. Pombe) (CDC25A))

Reactivity: Human, Mouse, Rat

Host: Mouse

Clonality: Monoclonal

Conjugate: This CDC25A antibody is un-conjugated

Application: Western Blotting (WB), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunoprecipitation (IP)

Clone: DCS-121

Product Details anti-CDC25A Antibody

Specificity: This antibody reacts with Human, Mouse and Rat CDC25A.

Cross-Reactivity (Details): Species reactivity (tested):Human, Mouse and Rat.

Characteristics: Synonyms: CDC-25A, M-phase inducer phosphatase 1, Dual specificity phosphatase Cdc25A, CellDivision Cycle 25A

Purification: Protein-A Sepharose Chromatography.

Immunogen: Full-length Human CDC25A fusion protein. Remarks: Hybridoma was established by fusion of Mouse myeloma cell NS-2 with Balb/cmouse splenocyte.

Isotype: IgG2a

Application Details

Application Notes: Western Blot: 1-5 μg/mLImmunoprecipitation: 3 μg/200-300 μL of cell extract. Immunohistochemistry: 1-5 μg/mLHeat treatment is necessary for Paraffin Embedded Sections. Microwave oven: 2 times for 10 minutes each in citrate buffer ( pH 6.5). Positive Controls: HeLa, Raji, NIH/3T3 and Rat-1 Cells. Detailed procedure is provided in Protocols.
Other applications not tested.
Optimal dilutions are dependent on conditions and should be determined by the user.

Protocol: SDS-PAGE & Western Blotting1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mMTris-HCl, pH 7. 2, 250 mM NaCl, 0. 1% NP-40, 2 mM EDTA, 10% glycerol) containingappropriate protease inhibitors. Incubate it at 4°C with rotating for 30 minutes, thensonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C and transfer the supernatant toanother tube. Measure the protein concentration of the supernatant and add the Lysisbuffer to make 8 mg/mL solution. 3) Mix the sample with equal volume of Laemmli’s sample buffer. 4) Boil the samples for 2 minutes and centrifuge. Load 10 μL of the sample per lane in a 1mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hourin a semi-dry transfer system. (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for the transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH7. 2) for 1 hour at room temperature, or overnight at 4°C. 7) Incubate the membrane with the anti-CDCC25A (DCS-121) monoclonal antibody (1-5μg/mL) diluted with 1% skimmed milk (in PBS, pH 7. 2) for 1 hour at room temperature. 8) Wash the membrane with PBS (5 minutes x 6 times). 9) Incubate the membrane with the 1: 10000 HRP-conjugated anti-mouse IgG diluted with1% skimmed milk (in PBS, pH 7. 2) for 1 hour at room temperature. 10) Wash the membrane with PBS (5 minutes x 6 times). 11) Wipe excess buffer from the membrane, then incubate it with appropriatechemiluminescence reagents for 1 minute. Remove extra reagent from the membrane bydabbing with a paper towel, and seal it in plastic wrap. 12) Expose to an X-ray film in a dark room for 5 minutes. Develop the film as usual. Theconditions for exposure and development may vary. Positive Controls for Western blotting: HeLa, Raji, NIH/3T3, Rat-1. Immunoprecipitation1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mMTris-HCl, pH 7. 2, 250 mM NaCl, 0. 1% NP-40, 2 mM EDTA, 10% glycerol) containingappropriate protease inhibitors. Incubate it at 4°C with rotating for 30 minutes, thensonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C and transfer the supernatant toanother tube. 3) Add 3 μg of the anti-CDC25A (DCS-121) monoclonal antibody into 250 μL of thesupernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4°C. Add 20μL of 50% Protein A-agarose beads resuspended in the Lysis buffer. Mix well and incubatewith gentle agitation for 60 minutes at 4°C. 4) Wash the beads 3-5 times with ice-cold Lysis buffer (centrifuge the tube at 2,500 x g for10 seconds). 5) Resuspend the beads in 20 μL of Laemmli’s sample buffer, boil for 3-5 minutes, andcentrifuge for 5 minutes. Use 10 μL/lane for the SDS-PAGE analysis. (See SDS-PAGE & Western blotting. )Positive Controls for immunoprecipitation: Raji cells. Immunohistochemical Staining for Paraffin-Embedded Sections: SAB method1) Deparaffinize the sections with Xylene 3 times for 3-5 minutes each.

Restrictions: For Research Use only

Handling

Concentration: 1.0 mg/mL

Buffer: PBS, pH 7.2 containing 50 % Glycerol without preservatives.

Preservative: Without preservative

Storage: -20 °C

Storage Comment: Store the antibody undiluted at -20 °C.
Shelf life: one year from despatch.

Expiry Date: 12 months

Target Details for CDC25A

Target: CDC25A (Cell Division Cycle 25 Homolog A (S. Pombe) (CDC25A))

Alternative Name: CDC25A

Background: The members of the CDC25 family, CDC25A, CDC25B, and CDC25C, activate the cyclin-dependent kinases at different points in the cell cycle by dephosphorylating key proteins. The ~65 kDa CDC25A protein is a tyrosine phosphatase that regulates the G1/S transition by activating cyclin E/Cdk2 and cyclin A/Cdk2 complexes, which are required for DNA synthesis. CDC25A acts as a checkpoint to prevent DNA replication following DNA damage. DNA damage induces phosphorylation of CDC25A, resulting in its rapid degradation via the ubiquitin-proteosome pathway, and thus silencing Cdk2 activity.Synonyms: CDC-25A, Cell Division Cycle 25A, Dual specificity phosphatase Cdc25A, M-phase inducer phosphatase 1

Gene ID: 993

UniProt: P30304

Pathways: Cell Division Cycle, Mitotic G1-G1/S Phases, M Phase