Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    Mono and Polyubiquitylated conjugates, mAb (FK2) HRP linked
    Catalogue Number:
    68-0122-025
    Size:
    25 µg
    Price:
    £125
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  • Species
    human
  • Source
    BALB/C Mouse implantation ascites
  • Quantity
    25 µg
  • Storage
    -20°C
  • Concentration
    1 mg/ml
  • Formulation
    10mM phosphate buffer, 0.15M NaCl PH7.4, 0.1% sodium azide
  • Molecular Weight
    150kDa
  • Stability
    12 months at -20°C; aliquot as required
  • Protein Sequence
    n/a
  • QA; Protein Identification
    n/a
  • QA; Activity
    Anti-Mono and Polyubiquitylated Conjugates mAb (FK2) HRP-linked Antibody Activity Assay: The activity of the anti-mono and polyubiquitylated conjugates mAb (FK2) HRP-linked antibody was tested for its ability to recognise mono and polyubiquitin conjugates (Lanes 2 and 3) but not free ubiquitin (Lane1). By Western blotting the specific recognition of mono and polyubiquitin conjugates by the antibody over free ubiquitin was demonstrated (Figure 1). In a priming and extension assay containing, UBE1 [6His-tagged] (Cat# 61-0001), UBE2W [6His-tagged] (Cat# 62-0085), UBE2N [untagged] (Cat# 62-0047), UBE2V1 [untagged] (Cat# 62-0059), Ubiquitin (Cat# 60-0001), CHIP [untagged] (Cat# 63-0003) and ATP. Using the anti mono and polyubiquitylated conjugates mAb (FK2) HRP-linked antibody, detection of polyubiquitin chains extending from mono-ubiquitylated CHIP (Lane 1) and free chains generated by UBE2N/UBE2V1 in the presence of CHIP (Lane 3) were observed. In the absence of CHIP, only the background detection of free polyubiquitin chains generated by UBE2N/UBE2V1 was observed (Lanes 5 and 7) (Figure 2).

Anti-mono and polyubiquitylated conjugates mAb (FK2) demonstrates specific recognition for monoubiquitylated and polyubiquitylated proteins but shows no reactivity with free ubiquitin (Fujimoro et al. 1994). The anti-polyubiquitylated conjugates mAb (FK2) has been extensively characterised and used not only to investigate ubiquitin chain formation on poly and mono ubiquitylated proteins by Western blotting but also in the detection of intracellular polyubiquitin chains in immunoassays (Takada et al. 1995; Fujimoro et al. 2005). The HRP linked anti-mono and polyubiquitylated conjugates mAb (FK2) facilitates the detection of mono and polyubiquitylated ubiquitin conjugates by Western blotting without the use of a secondary antibody.

References:
Fujimuro M, Sawada H, Yokosawa H (1994) Production and characterization of monoclonal antibodies specific to multiubiquitin chains of polyubiquitinated proteins. FEBS Lett 349 173-180.
Takada K, Nasu H, Hibi N, Tsukada Y, Ohkawa K, Fujimuro M, Sawada H, Yokosawa H (1995) Immunoassay for the quantification of intracellular multi-ubiquitin chains. Eur J Biochem 233 42-47.
Fujimuro M, Yokosawa H (2005) Production of antipolyubiquitin monoclonal antibodies and their use for characterization and isolation of polyubiquitinated proteins. Methods Enzymol 399 75-86.