The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/10000 - 1/50000. Detects a band of approximately 20 kDa (predicted molecular weight: 21 kDa).
1/10 - 1/100.
1/100 - 1/250. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
1/500 - 1/1000.
1/100 - 1/1000. ab172730-Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
FunctionAccepts the ubiquitin-like protein NEDD8 from the UBA3-NAE1 E1 complex and catalyzes its covalent attachment to other proteins. The specific interaction with the E3 ubiquitin ligase RBX1, but not RBX2, suggests that the RBX1-UBE2M complex neddylates specific target proteins, such as CUL1, CUL2, CUL3 and CUL4. Involved in cell proliferation.
PathwayProtein modification; protein neddylation.
Sequence similaritiesBelongs to the ubiquitin-conjugating enzyme family. UBC12 subfamily.
DomainBoth the N-terminal docking peptide and the catalytic core domain must bind the UBA3-NAE1 complex simultaneously for optimal transfer of NEDD8.
Overlay histogram showing Ramos cells stained with ab109507 (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 (ab109507, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Ramos cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
Western blot - UBE2M antibody [EPR5333] (ab109507)
All lanes : Anti-UBE2M antibody [EPR5333] (ab109507) at 1/10000 dilution
Lane 1 : 293T cell lysate Lane 2 : Ramos cell lysate