CDK6 antibody

Catalog No. ABIN487480

Product Details anti-CDK6 Antibody

Target: CDK6 (Cyclin-Dependent Kinase 6 (CDK6))

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This CDK6 antibody is un-conjugated

Application: Immunofluorescence (IF), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunoprecipitation (IP)

Specificity: This antibody react with Human Cdk6 on Immunoprecipitation, Immunohistochemistry and Immunocytochemistry. It is reported that DCS-130 recognizes the C-terminus of Human Cdk6 (300-326) (See Reference 3).

Cross-Reactivity (Details): Species reactivity (tested):Human.

Characteristics: Synonyms: PLSTIRE, Cell division protein kinase 6, Serine/threonine-protein kinase PLSTIRE

Purification: Protein-A Sepharose Chromatography.

Immunogen: Recombinant Human Cdk6. Remarks: Hybridoma was established by fusion of mouse myeloma cell NS-2 with Balb/cmouse splenocyte.

Clone: DCS-130

Isotype: IgG2a

Application Details

Application Notes: Immunocytochemistry: 10 μg/mLImmunoprecipitation: 10 μg/400 μL of cell extract from 5x10^6 cells. Positive Controls: HeLa, Jurkat cells. Immunohistochemistry: 10 μg/mLHeat treatment is necessary for Paraffin Embedded Sections. Microwave oven: 2 times for 10 minutes each in 10 mM citrate buffer ( pH 6.5)Positive Control: Tonsil. Detailed procedure is provided in Protocols.
Other applications not tested.
Optimal dilutions are dependent on conditions and should be determined by the user.

Protocol: 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 min at 4°C and transfer the supernatant toanother tube. 3) Add primary antibody as suggest in the APPLICATIONS into 400 µL of the supernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4oC. Add 20 ìL of 50%protein A Agarose resuspended in the cold Lysis buffer. Mix well and incubate with gentleagitation for 60 minutes at 4°C. 4) Wash the beads 3-5 times with the ice-cold Lysis buffer (centrifuge the tube at 2,500 x gfor 10 seconds). 5) Resuspend the beads in 20 μL of Laemmli’s sample buffer, boil for 3-5 min, andcentrifuge for 5 minutes. Use 10 μL/lane for the SDS-PAGE analysis. 6) Load 10 µL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel forelectrophoresis. 7) 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 precise transfer procedure. 8) 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. 9) Incubate the membrane with 1 µg/mL of anti-Cdk6 as primary antibody diluted with PBS,pH 7. 2 containing 1% skimmed milk for 1 hour at room temperature. (The concentration ofantibody will depend on condition. )10) Wash the membrane with PBS-T [0. 05% Tween-20 in PBS] (5 minutes x 3 times). 11) Incubate the membrane with the 1: 10,000 HRP-conjugated anti-mouse IgG diluted with1% skimmed milk (in PBS, pH 7. 2) for 1 hour at RT. 12) Wash the membrane with PBS-T (5 minutes x 6 times). 13) Wipe excess buffer on the membrane, then incubate it with appropriatechemiluminescence reagent for 1 minute. Remove extra reagent from the membrane bydabbing with paper towel, and seal it in plastic wrap. 14) Expose to an X-ray film in a dark room for 5 minutes. Develop the film as usual. Thecondition for exposure and development may varyPositive Control for Immunoprecipitation: HeLa, Jurkat. Immunohistochemical Staining for Paraffin-Embedded Sections: SAB method1) Deparaffinize the sections with Xylene 3 times for 3-5 minutes each. 2) Wash the slides with Ethanol 3 times for 3-5 minutes each. 3) Wash the slides with PBS 3 times for 3-5 minutes each. 4) Heat treatmentHeat treatment by Microwave: Place the slides put on staining basket in 500 mL beakerwith 500 mL of 10 mM citrate buffer (pH 6. 5). Cover the beaker with plastic wrap, then

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 CDK6

Target: CDK6 (Cyclin-Dependent Kinase 6 (CDK6))

Alternative Name: CDK6 (CDK6 Products)

Synonyms: CDK6 antibody, PLSTIRE antibody, 5830411I20 antibody, AI504062 antibody, Crk2 antibody, cyclin dependent kinase 6 antibody, cyclin-dependent kinase 6 antibody, CDK6 antibody, Cdk6 antibody

Background: Cyclin-dependent kinase 6 (Cdk6) is a 38-40 kDa member of the Cdk family of mitotic kinases involved in cell cycle progression. The Cdk6 phosphoprotein, like Cdk4, associates with D-cyclins to regulate cell proliferation. Following translocation to the nucleus, the Cdk6 is phosphorylated by Cdk-Activating Kinase (CAK), and the Cdk6 complex, in association with PCNA, phosphorylates the retinoblastoma (Rb) protein. Overexpression of Cdk6 has been associated with several different types of cancer.Synonyms: Cell division protein kinase 6, PLSTIRE, Serine/threonine-protein kinase PLSTIRE

Gene ID: 1021

UniProt: Q00534

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