The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 - 5 µg/ml. Predicted molecular weight: 65 kDa.
Use at an assay dependent concentration. PubMed: 20725033
Use 1µg for 106 cells. ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
Use a concentration of 1.5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
FunctionE3 ubiquitin-protein ligase required for accumulation of repair proteins to sites of DNA damage. Acts with UBE2N/UBC13 to amplify the RNF8-dependent histone ubiquitination. Recruited to sites of DNA damage at double-strand breaks (DSBs) by binding to ubiquitinated histone H2A and ubiquitinates histone H2A and H2AX, leading to amplify the RNF8-dependent H2A ubiquitination and promoting the formation of 'Lys-63'-linked ubiquitin conjugates. This leads to concentrate ubiquitinated histones H2A and H2AX at DNA lesions to the threshold required for recruitment of TP53BP1 and BRCA1.
PathwayProtein modification; protein ubiquitination.
Involvement in diseaseDefects in RNF168 are the cause of Riddle syndrome (RIDDLES) [MIM:611943]. Riddle syndrome is characterized by increased radiosensitivity, immunodeficiency, mild motor control and learning difficulties, facial dysmorphism, and short stature. Defects are probably due to impaired localization of TP53BP1 and BRCA1 at DNA lesions.
Sequence similaritiesBelongs to the RNF168 family. Contains 1 RING-type zinc finger.
DomainThe MIU motifs (motif interacting with ubiquitin) mediate the interaction with ubiquitin and the localization at sites of DNA damage.
Cellular localizationNucleus. Localizes to sites of DNA damage.
Immunofluorescence analysis of HepG2 cells labelling RNF168 with ab58063 at 10μg/ml.
Flow Cytometry - Anti-RNF168 antibody (ab58063)
Overlay histogram showing HepG2 cells stained with ab58063 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab58063, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 80% methanol (5 min) /permeabilized in 0.1% PBS-Tween used under the same conditions.
Zhu B et al. K63-linked ubiquitination of FANCG is required for its association with the Rap80-BRCA1 complex to modulate homologous recombination repair of DNA interstand crosslinks. OncogeneN/A:N/A (2014).
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