p53 antibody (pSer315)

Catalog No. ABIN487474

Product Details anti-p53 Antibody

Target: p53 (TP53) (Tumor Protein P53 (TP53))

Binding Specificity: pSer315

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This p53 antibody is un-conjugated

Application: Western Blotting (WB), Immunoprecipitation (IP)

Specificity: This antibody reacts with Human p53 phosphorylated at Ser315.

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

Characteristics: Synonyms: Cellular tumor antigen p53, Tumor suppressor p53, Phosphoprotein p53, NY-CO-13

Purification: Protein-A Sepharose Chromatography

Immunogen: A 15-amino acid phosphopeptideAA Sequence: RALPNNTSSpSPQPKKKbRemarks: Hybridoma was established by fusion of mouse myeloma cell SP-2 with Balb/cmouse splenocytes.

Clone: FPS315

Isotype: IgG1

Application Details

Application Notes: Immunoprecipitation/Western blotting: 5 μg/mLPositive Control: Raji cells. Detailed procedure is provided in Protocols. This monoclonal antibody should be used by Western blot in combination withImmunoprecipitation with anti-p53 (DO-1). This antibody is not recommended for use by Western blotting using crude cell lysates.
Other applications not tested.
Optimal dilutions are dependent on conditions and should be determined by the user.

Protocol: Immunoprecipitation / SDS PAGE & Western Blotting1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50mM Tris-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 100 µL of the anti-p53 monoclonal antibody into 200 µL of the supernatant. Mix welland incubate with gentle agitation for 30-120 minutes at 4°C. Add 20 µL of 50% ProteinA-agarose beads resuspended in the Lysis buffer. Mix well and incubate with gentleagitation 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 ìL of Laemmli’s sample buffer, boil for 3-5 minutes, andcentrifuge for 5 minutes. Load 10 µL of the sample per lane in a 1 mm thickSDS-polyacrylamide gel for electrophoresis. 6) 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 specific transfer procedure. 7) 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. 8) Incubate the membrane with the anti-phospho p53 Ser315 monoclonal antibody (5µg/mL) diluted with 1% skimmed milk (in PBS, pH 7. 2) for 1 hour at room temperature. 9) Wash the membrane with PBS (5 minutes x 6 times). 10) Incubate the membrane with the 1: 10000 POD-conjugated anti-mouse IgG ê dilutedwith 1% skimmed milk (in PBS, pH 7. 2) for 1 hour at room temperature. 11) Wash the membrane with PBS (5 minutes x 6 times)12) 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. 13) Expose to 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 Immunoprecipitation/Western blotting: Raji 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: 4 °C

Storage Comment: Store the antibody undiluted at 2-8 °C.
Shelf life: one year from despatch.

Expiry Date: 12 months

Target Details for p53

Target: p53 (TP53) (Tumor Protein P53 (TP53))

Alternative Name: p53 (TP53) (TP53 Products)

Background: P53 tumor suppressor proteins play a critical role in cellular defense against DNA-damaging reagents. Activation of p53 causes cell cycle arrest and sometimes, apoptosis, resulting in the prevention of genetically damaged cells from proliferating. DNA damage increases p53 protein levels and activates transcription of the p21 gene. The p21 protein binds to and inhibits the cell cycle regulator cdc2 kinase, causing G1 arrest. p53 is regulated by phosphorylation at multiple sites by several different protein kinases. The cdc2 kinase phosphorylates Ser315 of human p53 in vitro, and the majority of p53 protein is phosphorylated at Ser315 following irradiation damage.Synonyms: Cellular tumor antigen p53, NY-CO-13, Phosphoprotein p53, Tumor suppressor p53

Gene ID: 7157

UniProt: P04637

Pathways: p53 Signaling, MAPK Signaling, PI3K-Akt Signaling, Apoptosis, AMPK Signaling, Chromatin Binding, ER-Nucleus Signaling, Positive Regulation of Endopeptidase Activity, Hepatitis C, Protein targeting to Nucleus, Autophagy, Warburg Effect