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Arginylation (N-Term) anticorps

DB Hôte: Lapin Polyclonal unconjugated
N° du produit ABIN4368250
  • Antigène
    Arginylation
    Épitope
    N-Term
    Reactivité
    Veuillez nous consulter SVP
    Hôte
    • 23
    Lapin
    Clonalité
    • 23
    Polyclonal
    Conjugué
    • 3
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    Inconjugué
    Application
    • 23
    • 11
    Dot Blot (DB)
    Specificité
    N-terminal arginylation
    Purification
    Affinity purified serum. Antibodies are purified using substactive purification method.
    Immunogène
    KLH-conjugated synthetic peptide: H-RDHKHANQHMSVC-NH2
  • Indications d'application
    1 : 1000 (Dot)
    Restrictions
    For Research Use only
  • Validation #100047 (Western Blotting)
    'Independent Validation' signe
    by
    Leibniz-Institut für Pflanzenbiochemie (IPB), Halle
    No.
    #100047
    Date
    02.09.2016
    Antigène
    Arginylation (N-Term)
    Numéro du lot
    Application validée
    Western Blotting
    Contrôle positif
    Arg-Asp-starting recombinant protein
    Contrôle négative
    Asp-starting recombinant protein
    Conclusion
    ABIN4368250 specifically detects recombinant protein with the N-terminal amino acid sequence Arg-Asp in a Western blot.
    'Independent Validation' signe
    Validation Images
    Protocole
    Anticorps primaire
    ABIN4368250
    Anticorps secondaire
    goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004)
    Full Protocol
    • Recombinant protein:
      • Bacterial protein expression and purification according to Naumann et al. 2016.
      • 1-15µg total protein were mixed with SDS-loading buffer and boiled for 5min.
    • Plant extracts:
      • Plant material (3 weeks old Arabidopsis) was ground with liquid nitrogen in RIPA-buffer and incubated for 15min at 4°C.
      • Debris was centrifuged at max. speed for 10min. Total protein content of the supernatant was determined.
      • 23.6µg (first experiment) or 94µg (second experiment) respectively were mixed with SDS-loading buffer and boiled for 5min.
    • Proteins were separated on a 12% denaturing SDS-PAGE gel (Carl Roth) in a Bio-Rad Tetra cell and transferred in a Bio-Rad semidry-blot apparatus for 1.5h at 1mA/cm2 to PVDF membrane (GE Healthcare).
    • The membrane was blocked in TBST with 5% milk powder (Carl Roth) for 1h in at RT.
    • Incubation with primary antibody ABIN4368250 (antibodies-online) diluted 1:1000 in TBST with 3% milk powder overnight at 4°C.
    • Washing with TBST with 3% milk powder for 3x 5min.
    • Incubation with secondary antibody goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) diluted 1:2500 in TBST with 3% milk powder for 1h at RT.
    • Washing with TBST with 3% milk powder for 5x 5min.
    • Detection with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) on a standard X-ray film; exposure 30s.
    Notes
    ABIN4368250 recognizes the recombinant protein with N-terminal Arg-Asp shown in the image. In case of a different recombinant protein (not shown) the antibody did not only recognize the protein with the N-terminal sequence Arg-Asp but also with Asn or Asp at the amino-terminus.
  • Validation #100046 (Protein Array)
    'Independent Validation' signe
    by
    Leibniz-Institut für Pflanzenbiochemie (IPB), Halle
    No.
    #100046
    Date
    23.08.2016
    Antigène
    Arginylation (N-Term)
    Numéro du lot
    Application validée
    Protein Array
    Contrôle positif
    N-terminally arginylated synthetic peptides REHKHANQHMSVC, RDELPESIDWRKKGAV
    Contrôle négative
    un-arginylated synthetic peptides HKHRDANQHMSVC, HKHREANQHMSVC, HKHANQHMSVA
    Conclusion
    The antibody specifically recognizes peptides with the N-terminal amino acid sequence RD and to a lesser extent peptides with the N-terminal sequence RE.
    'Independent Validation' signe
    Validation Images
    Protocole
    Anticorps primaire
    ABIN4368250
    Anticorps secondaire
    goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004)
    Full Protocol
    • Peptide array synthesis according to Yim et al. 2015.
    • Block membrane with TBST with 7.5% milk powder overnight at 4°C.
    • Incubation with primary N-terminal arginylation antibody (antibodies-online, ABIN4368250) diluted 1:1000 in TBST with 3% milk powder for 1h at RT.
    • Washing with TBST for 3x 10min.
    • Transfer proteins onto PVDF membrane in a semi-dry for 30min at 0.8mA/cm2.
    • Block the membrane with TBST with 4% milk powder ON at 4°C.
    • Incubation with secondary antibody goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) diluted 1:2500 in TBST with 3% milk powder for 1h at RT.
    • Wash membrane 5x 20min in TBST.
    • Chemiluminescence detection with ECL (Thermo Scientific pico kit) on a standard X-ray film; exposure 30s.
    Notes
  • Format
    Lyophilized
    Reconstitution
    For reconstitution add 25 μL of sterile water to each tube.
    Buffer
    PBS pH 7.4
    Stock
    -20 °C
    Stockage commentaire
    Store lyophilized/reconstituted at -20°C, once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
  • Antigène
    Arginylation
    Classe de substances
    Chemical
    Sujet
    Arginylation is a post-translational modification of an existing peptide chain by addition of an extra arginine. This modification changes primary sequence of protein as well as it's surface change. It is mediated by arginyltransferase ATE1. Arginylation plays an essential role in multiple physiological pathways, for example, in vivo arginylation constitutes a mechanism for degradation of preprocessed proteins or proteolytic fragments that bear Asp and Glu on their N-termini.
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