The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
ChIP: Use at an assay dependent dilution (PMID 19237537.
ICC/IF: Use at a concentration of 1 µg/ml.
WB: 1/250. Detects a band of approximately 116 kDa (predicted molecular weight: 119 kDa).
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
E3 ubiquitin-protein ligase component of the LUBAC complex which conjugates linear polyubiquitin chains in a head-to-tail manner to substrates and plays a key role in NF-kappa-B activation and regulation of inflammation. LUBAC conjugates linear polyubiquitin to IKBKG and RIPK1 and is involved in activation of the canonical NF-kappa-B and the JNK signaling pathways. Linear ubiquitination mediated by the LUBAC complex interferes with TNF-induced cell death and thereby prevents inflammation. LUBAC is proposed to be recruited to the TNF-R1 signaling complex (TNF-RSC) following polyubiquitination of TNF-RSC components by BIRC2 and/or BIRC3 and to conjugate linear polyubiquitin to IKBKG and possibly other components contributing to the stability of the complex. Binds polyubiquitin of different linkage types.
Expressed in both normal and transformed breast epithelial cell lines.
ICC/IF image of ab46322 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab46322, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% formaldehyde fixed (10 min) Hek293 cells at 5µg/ml.
Western blot - Anti-RNF31/HOIP antibody - ChIP Grade (ab46322)This image is courtesy of an anonymous Abreview
Western blot analysis of A549 cell lysate (100μg/lane) RNF31/HOIP with ab46322 at 1/1000. A HRP-conjugated Goat anti-rabbit polyclonal (1/10000) was used as the secondary antibody.