CDC25A Antikörper

Produktnummer ABIN487337

Der Maus Monoklonal anti-CDC25A Antikörper wird verwendet zum Nachweis von CDC25A in Proben von Human, Maus und Ratte. Er wurde validiert für WB, IHC (p) und IP.

Kurzübersicht für CDC25A Antikörper (ABIN487337)

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

Reaktivität: Human, Maus, Ratte

Wirt: Maus

Klonalität: Monoklonal

Konjugat: Dieser CDC25A Antikörper ist unkonjugiert

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

Klon: DCS-121

Produktdetails anti-CDC25A Antikörper

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

Kreuzreaktivität (Details): Species reactivity (tested):Human, Mouse and Rat.

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

Aufreinigung: 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.

Isotyp: IgG2a

Anwendungsinformationen

Applikationshinweise: 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.

Protokoll: 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.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Konzentration: 1.0 mg/mL

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

Konservierungsmittel: Without preservative

Lagerung: -20 °C

Informationen zur Lagerung: Store the antibody undiluted at -20 °C.
Shelf life: one year from despatch.

Haltbarkeit: 12 months

Details zu CDC25A

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

Andere Bezeichnung: CDC25A

Hintergrund: 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

Gen-ID: 993

UniProt: P30304

Pathways: Zellzyklus, Mitotic G1-G1/S Phases, M Phase