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p53 anticorps (meLys370)

TP53 Reactivité: Humain WB, ELISA Hôte: Lapin Polyclonal unconjugated
N° du produit ABIN4902067
  • Antigène Voir toutes p53 (TP53) Anticorps
    p53 (TP53) (Tumor Protein P53 (TP53))
    Épitope
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    meLys370
    Reactivité
    • 900
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    Humain
    Hôte
    • 677
    • 239
    • 6
    • 2
    • 1
    • 1
    Lapin
    Clonalité
    • 611
    • 314
    • 1
    Polyclonal
    Conjugué
    • 518
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    Cet anticorp p53 est non-conjugé
    Application
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    Western Blotting (WB), ELISA
    Fonction
    Rabbit polyclonal to p53 (Mono Methyl Lys370).
    Specificité
    Mono-Methyl-p53 (K370) Polyclonal Antibody detects endogenous levels of p53 around the methylation site of K370 protein.
    Attributs du produit
    Rabbit Polyclonal to p53 (Mono Methyl Lys370).
    Purification
    The antibody was affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogen.
    Immunogène
    Synthesized peptide derived from human p53 around the methylation site of K370.
    Isotype
    IgG
    Top Product
    Discover our top product TP53 Anticorps primaire
  • Indications d'application
    WB 1:500-1:2000
    ELISA 1:10000
    Commentaires

    Ubiquitous. Isoforms are expressed in a wide range of normal tissues but in a tissue-dependent manner. Isoform 2 is expressed in most normal tissues but is not detected in brain, lung, prostate, muscle, fetal brain, spinal cord and fetal liver. Isoform 3 is expressed in most normal tissues but is not detected in lung, spleen, testis, fetal brain, spinal cord and fetal liver. Isoform 7 is expressed in most normal tissues but is not detected in prostate, uterus, skeletal muscle and breast. Isoform 8 is detected only in colon, bone marrow, testis, fetal brain and intestine. Isoform 9 is expressed in most normal tissues but is not detected in brain, heart, lung, fetal liver, salivary gland, breast or intestine.

    Restrictions
    For Research Use only
  • Validation #101268 (Western Blotting)
    'Independent Validation' signe
    by
    Department of Prevention and Therapy of Chronic Diseases, Institute for Advanced Biosciences, CNRS UMR5309
    No.
    #101268
    Date
    03.08.2017
    Antigène
    p53K370me
    Numéro du lot
    B1001
    Application validée
    Western Blotting
    Contrôle positif

    p53, in vitro specifically p53K370me1 methylated using methyltransferase SMYD2

    ectopic SMYD-methylated wildtype p53 from 293T cell extracts

    Contrôle négative

    p53, in vitro methylated using methyltransferases G9a and SETD8

    p53ΔCTD, lacking C-terminal domain containing known methylable lysines

    p53K370R, lacking the lysine 370 methylation site, mutated into arginine

    Conclusion

    Passed. ABIN4902067 specifically recognizes monomethylated p53K370me1 and to a lesser extent p53Kme1.

    'Independent Validation' signe
    Validation Images
    Protocole
    Anticorps primaire
    ABIN4902067
    Anticorps secondaire
    HRP-conjugated goat anti-rabbit or anti-mouse antibody (Biorad)
    Full Protocol
    • In vitro p53 methylation assay:
      • Clone SMYD2 and p53 from human cDNA library into pGex6p1 vector.
      • Perform p53 mutagenesis using QuickChange II site-directed mutagenesis kit (Agilent, 200523).
      • Transform vectors into BL21 pLys bacteria (Agilent) and induced protein expression with 0.1mM IPTG in 250mL culture ON at 19°C.
      • Purify proteins by GST pulldown and quantify using Bradford assay and Coomassie gel staining.
      • For the in vitro methylation assays incubate 1 to 2µg recombinant protein with 1µg of recombinant methyltransferases and 2µCi 3H-AdoMet (American Radiolabelled Chemicals) in buffer containing 50mM Tris-HCl pH8.0, 10% glycerol, 20mM KCl, 5mM MgCl2, and 1mMPMSF at ON at 30°C.
      • Resolve the reaction mixture by SDS–PAGE, followed by autoradiography and Coomassie stain (Pierce).
    • "In cellulo” p53 methylation assay:
      • Subclone SMYD2 and p53 constructs from pGex6p1 vectors into pcagFLAG (SMYD2) or pCDNA3.1HA (p53).
      • Transfect plasmids into 293T cells using Lipofectamine 2000 (ThermoFisher) according to supplier’s protocol.
      • Harvest cells 36h post-transfection to allow methylation of ectopic p53 by SMYD2 in cells.
    • Western blot:
      • Separate 20µg of each sample on freshly cast SDS-PAGE gel in a gel electrophoresis chamber (BioRad) for 1h at 140V and RT.
      • Transfer proteins onto ethanol-activated 0.2µm Hybond PVDF membrane (Amersham) with a semi-dry Trans-Blot Turbo (BioRad) for 30min at 20V.
      • Block the membrane using 5% milk in TBS-Tween 0.1% for 1h at RT.
      • Incubate the membrane with primary rabbit anti-p53 meLys370 antibody (antibodies-online, ABIN4902067, LOT B1001), mouse anti-HA antibody (Sigma), or mouse anti-Flag tag antibody (Sigma) diluted 1:1000 in 1% BSA TBS-Tween 0.1% ON at 4°C.
      • Wash the membrane 3x for 10min in TBS-Tween 0.1%.
      • Incubate the membrane with secondary HRP-conjugated goat anti-rabbit or anti-mouse antibody (Biorad) diluted 1:3000 in 1% BSA TBS-Tween 0.1% for 1h at RT.
      • Wash the membrane 3x for 10min in TBS-Tween 0.1%.
      • Visualize protein bands using ECL RevelBlot intense (Ozyme) and signal detection on Hyperfilm ECL 18 x24cm (Amersham).
    Notes
    • Antibody ABIN4902067 recognizes greatly its specific substrate in vitro: it shows a strong signal when SMYD2 is present, implying monomethylation of K370. The signal is lost when using p53K370R and p53ΔCTD mutants. However, ABIN4902067 also recognizes to a lesser extent in vitro other p53 methylation such as p53 K373me1/2 and p53K382me2 caused by G9a and SETD8 respectively.
    • The antibody does recognize unmethylated p53 when using a large quantity of the recombinant protein (not shown).
    • ABIN4902067 gives a very clean and specific signal when using ectopic p53 from 293T cell extract: only p53 methylated by SMYD2 is detected, not the p53K370R mutant. The antibody can even be used without an initial immunoprecipitation of total p53 as observable in our figure.
    • The binding affinity of ABIN4902067 can be ordered as follows: p53K370me1>p53Kme1>>>p53.
  • Format
    Liquid
    Concentration
    1 mg/mL
    Buffer
    Liquid in PBS containing 50 % glycerol, 0.5 % BSA and 0.02 % sodium azide.
    Agent conservateur
    Sodium azide
    Précaution d'utilisation
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Conseil sur la manipulation
    Avoid repeated freeze/thaw cycles.
    Stock
    -20 °C
    Stockage commentaire
    Store at -20°C, and avoid repeat freeze-thaw cycles.
  • Antigène
    p53 (TP53) (Tumor Protein P53 (TP53))
    Autre désignation
    p53 (TP53 Produits)
    Synonymes
    anticorps BCC7, anticorps LFS1, anticorps P53, anticorps TRP53, anticorps p53, anticorps Trp53, anticorps brp53, anticorps drp53, anticorps etID22686.5, anticorps fb40d06, anticorps wu:fb40d06, anticorps zgc:111919, anticorps Tp53, anticorps bbl, anticorps bfy, anticorps bhy, anticorps p44, anticorps tp53, anticorps Xp53, anticorps TP53, anticorps tumor protein p53, anticorps cellular tumor antigen p53, anticorps transformation related protein 53, anticorps tumor protein p53 L homeolog, anticorps TP53, anticorps CpipJ_CPIJ002758, anticorps Tp53, anticorps tp53, anticorps Trp53, anticorps tp53.L
    ID gène
    7157
    UniProt
    P04637
    Pathways
    Signalisation p53, Signalisation MAPK, Signalisation PI3K-Akt, Apoptose, AMPK Signaling, Chromatin Binding, ER-Nucleus Signaling, Positive Regulation of Endopeptidase Activity, Hepatitis C, Protein targeting to Nucleus, Autophagy, L'effet Warburg
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