CDKN2D Antikörper

Produktnummer ABIN487338

Produktdetails anti-CDKN2D Antikörper

Target: CDKN2D (Cyclin-Dependent Kinase Inhibitor 2D (p19, Inhibits CDK4) (CDKN2D))

Reaktivität: Human

Wirt: Maus

Klonalität: Monoklonal

Konjugat: Dieser CDKN2D Antikörper ist unkonjugiert

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

Spezifität: This antibody reacts with Human p19INK4d

Kreuzreaktivität (Details): Species reactivity (tested):Human.

Produktmerkmale: Synonyms: p19-INK4d, p19, INK4D, Cyclin-dependent kinase 4 inhibitor D

Aufreinigung: Protein-A Sepharose Chromatography.

Immunogen: Bacterially produced GST-tagged fusion proteins of full-length p19INK4d. Remarks: Hybridoma was established by fusion of Mouse myeloma cell NS-2 with Balb/cmouse splenocyte.

Klon: DCS-100

Isotyp: IgG1

Anwendungsinformationen

Applikationshinweise: Western Blot: 1 μg/mLPositive Control: Jurkat cells. Immunoprecipitation: 3 μg/200-300 μL of cell extract. Positive Control: Jurkat cells. 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 Control: Tonsil Tissue. 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-p19INK4d (DCS-100) monoclonal antibody (1μg/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 Control for Western blotting: Jurkat Cells. 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-p19INK4d (DCS-100) 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: Jurkat cells.

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 CDKN2D

Target: CDKN2D (Cyclin-Dependent Kinase Inhibitor 2D (p19, Inhibits CDK4) (CDKN2D))

Andere Bezeichnung: CDKN2D / p19INK4d (CDKN2D Produkte)

Hintergrund: The INK4 family of proteins consists of four members that block progression from the G(1)-to-S phase of the cell cycle by inhibiting the activity of Cdk4 and Cdk6. p19INK4d is a 165 amino acid protein with strong structural and functional similarity to p16INK4a, a known tumor suppressor. Mutations in p19INK4d have also been associated with human osteosarcomas.Synonyms: Cyclin-dependent kinase 4 inhibitor D, INK4D, p19, p19-INK4d

Gen-ID: 1032

UniProt: P55273

Pathways: Zellzyklus, Sensory Perception of Sound, Mitotic G1-G1/S Phases, Negative Regulation of intrinsic apoptotic Signaling