RNF8 [GST-tagged]


Catalogue Number
63-0021-025
Product Size
25 µg
Price £
£175
Accession Number
NP_003949
Residues Expressed
1-485
Certificate of Analysis Size
25 µg
Species
Human
Source
E. coli expression
Quantity
25 μg
Storage
-70°C
Concentration
0.5 mg/ml
Formulation
50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
Molecular Weight
~83.4 kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: NP_003949. For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

E3 ligase assay: The ubiquitin conjugating activity of GST-RNF8 was validated through its ability to catalyse the generation of polyubiquitin chains in the presence of the E1 activating enzyme His-UBE1, the E2 conjugating enzyme His-UBE2D4 (UbcH5d) (several E2s were tested, data generated with this E2 is provided by way of example) and ubiquitin. Incubation of GST-RNF8 for 30 minutes at 30°C in the presence of ubiquitin, His-UBE1, His-UBE2D4 and ATP (Lane 1) was compared alongside two control reactions with either ATP (Lane 2) or GST-RNF8 (Lane 3) excluded from the reaction. Ubiquitin conjugates were identified by Western blotting using an anti-ubiquitin conjugate antibody and these were observed only in the presence of both ATP and GST-RNF8.


Background

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including the regulated and targeted proteasome-dependent degradation of substrate proteins. Three classes of enzymes are involved in the process of ubiquitylation; activating enzymes (E1s), conjugating enzymes (E2s) and protein ligases (E3s). Ring Finger Protein 8 (RNF8) is a member of the E3 protein ligase family and cloning of the human gene was first described by Ishikawa et al. (1998). RNF8 is required for the ubiquitylation of some nuclear proteins, promoting their subsequent degradation (Kolas et al., 2007). RNF8 has also been shown to interact with the E2 conjugating enzyme Ubc13 (UBE2N) recruiting BRAC1 and 53BP1 to sites of nuclear damage (Kolas et al., 2007; Lok et al., 2011; Santos et al., 2010). RNF8 knockout mice display growth retardation and an increased pre-disposition to cancer (Li et al., 2010).


References

Ishikawa K, Nagase T, Suyama M, Miyajima N, Tanaka A, Kotani H, Nomura N, Ohara O (1998) Prediction of the coding sequences of unidentified human genes. X. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro. DNA Res 5, 169-76.

Kolas NK, Chapman JR, et al. (2007) Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science 318, 1637-40.

Li L, Halaby MJ, et al. (2010) Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer. J Exp Med 207, 983-97.

Lok GT, Sy SM, Dong SS, Ching YP, Tsao SW, Thomson TM, Huen MS (2011) Differential regulation of RNF8-mediated Lys48- and Lys63-based poly-ubiquitylation. Nucleic Acids Res. [Epub ahead of print]

Santos MA, Huen MS, et al. (2010) Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8. J Exp Med 207, 973-81.