Probable E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. May be required for development of the head mesenchyme and neural tube closure.
ab101992 stained HeLa cells. The cells were 100% methanol fixed (5 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 ab101992 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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.
Western blot - HECTD1 antibody (ab101992)
All lanes : Anti-HECTD1 antibody (ab101992) at 0.1 µg/ml
Lane 1 : HeLa whole cell lysate at 50 µg Lane 2 : HeLa whole cell lysate at 15 µg Lane 3 : HeLa whole cell lysate at 5 µg Lane 4 : 293T whole cell lysate at 50 µg
Developed using the ECL technique
Predicted band size : 290 kDa
Exposure time : 3 minutes
Immunoprecipitation - HECTD1 antibody (ab101992)
ab101992 at 1 µg/ml staining HECTD1 in HeLa cell lysate immunoprecipitated using ab101992 at 3 µg/mg lysate (1 mg/IP; 20% of IP loaded/lane).
Detection: Chemiluminescence with exposure time of 3 minutes