p21 antibody

Catalog No. ABIN487485

The Mouse Monoclonal anti-p21 antibody (Clone DCS-60) (ABIN487485) specifically detects p21 in WB, IHC (p) and IP. The antibody is reactive with Human samples.

Quick Overview for p21 antibody (ABIN487485)

Target: p21 (CDKN1A) (Cyclin-Dependent Kinase Inhibitor 1A (p21, Cip1) (CDKN1A))

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This p21 antibody is un-conjugated

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

Clone: DCS-60

Product Details anti-p21 Antibody

Specificity: This antibody reacts with Human p21WAF1/CIP1.

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

Characteristics: Synonyms: CAP20, CDKN1, CIP1, MDA6, MDA-6, PIC1, SDI1, WAF1, Cyclin-dependent kinase inhibitor 1,CDK-interacting protein 1, Melanoma differentiation-associated protein 6

Purification: Protein-A Sepharose Chromatography.

Immunogen: Full-length Recombinant Human p21WAF1/CIP1. 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/mLPositive Control: HeLa Cells. Immunoprecipitation: 3 μg/200-300 μL of cell extract. Positive Control: HeLa 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 Controls: 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.

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-p21WAF1/CIP1 (DCS-60) 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 POD-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: HeLa. 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-p21WAF1/CIP1 (DCS-60) 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: HeLa cells.

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 p21

Target: p21 (CDKN1A) (Cyclin-Dependent Kinase Inhibitor 1A (p21, Cip1) (CDKN1A))

Alternative Name: CDKN1A / p21WAF1

Background: P21WAF1/CIP1 is one member of the CIP/KIP family that controls the cell cycle by inhibiting cyclin dependent kinases (CDKs) activity. Increased expression of p21WAF1/CIP1 may play an important role in the growth arrest induced in transformed cells. It is reported that hypermethylation of the p21WAF1/CIP1 promoter region inactivate p21WAF1/CIP1 gene leading tumorigenesis, and also reported that p21WAF1/CIP1 acts as an inhibitor of apoptosis in a number of systems in addition to being an inhibitor of cell proliferation.Synonyms: CAP20, CDK-interacting protein 1, CDKN1, CIP1, Cyclin-dependent kinase inhibitor 1, MDA-6, MDA6, Melanoma differentiation-associated protein 6, PIC1, SDI1, WAF1

Gene ID: 1026

UniProt: P38936

Pathways: p53 Signaling, PI3K-Akt Signaling, Cell Division Cycle, AMPK Signaling, Fc-epsilon Receptor Signaling Pathway, EGFR Signaling Pathway, Neurotrophin Signaling Pathway, Mitotic G1-G1/S Phases, DNA Replication, Hepatitis C, Synthesis of DNA, Autophagy