Mono and Polyubiquitylated conjugates, mAb (FK2) Biotin linked


Catalogue Number
68-0123-025
Product Size
25 µg
Price £
£125
Accession Number
n/a
Residues Expressed
n/a
Alternate Product Size
100 µg
Certificate of Analysis Size
25 µg
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 -70°C; aliquot as required
Protein Sequence
n/a
QA; Protein Identification
n/a
QA Activity

Anti-Mono and Polyubiquitylated Conjugates mAb (FK2) Biotin-linked Antibody Activity Assay:The activity of the anti-mono and polyubiquitylated conjugates mAb (FK2) biotin-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 antimono and polyubiquitylated conjugates mAb (FK2) biotin-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).


Background

The 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-mono and polyubiquitylated conjugates mAb (FK2) has been extensively characterised and used not only to investigate ubiquitin chain formation on mono and polyubiquitylated proteins by Western blotting but also in the detection of intracellular polyubiquitin chains in immunoassays (Takada et al. 1995; Fujimoro et al. 2005). The biotin linked anti-mono and polyubiquitylated conjugates mAb (FK2) facilitates the detection of mono and polyubiquitylated conjugates with the use of a streptavidin HRP conjugate.


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.