||Recombinant Human Ubiquitin Conjugating Enzyme 12 produced inE.coliis a single non-glycosylated polypeptide chain containing 216 amino acids and having a molecular mass of approximately 25 kDa.
||UbcH12 is functional inin vitroNEDDylation reactions. It has been shown to form a thioester linkage with NEDD8 in the presence of the NEDD8 activating enzyme complex Uba3/APP-BP1. APP-BP1 binds to the amyloid precursor protein (APP) carboxy terminal domain and is important in conjunction with Uba3 and UbcH12 in driving cells through the S to M checkpoint. It was demonstrated to be the E2 responsible for the NEDDylation of the Cul-1 component of the SCF(β-TRCP) complex which is important as the E3-ligase in the ubiquitinylation of IκBα. NEDDylation of Cul-1 is essential for conjugation and processing of NF-κB p105 by SCF(β-TRCP) following phosphorylation of the complex. A dominant negative form of UbcH12, previously demonstrated to sequester NEDD8 and inhibit its conjugation, inhibits both conjugation and processing of p105, which is alleviated by wild-type UbcH12.
||>95% by SDS-PAGE and HPLC analyses.
||Lyophilized from a 0.2μm filtered concentrated (1mg/ml) solution in 1×PBS, 1mM DTT, pH 7.5.
||Less than 1EU/μg of rHuUbcH12 as determined by LAL method.
||We recommend that this vial be briefly centrifuged prior to opening to bring the contents to the bottom. Reconstitute in sterile distilled water or aqueous buffer containing 0.1% BSA to a concentration of 0.1-1.0 mg/mL. Stock solutions should be apportioned into working aliquots and stored at <-20oC. Further dilutions should be made in appropriate buffered solutions.
||This lyophilized preparation is stable at 2-8oC, but should be kept at -20oC for long term storage, preferably desiccated. Upon reconstitution, the preparation is stable for up to one week at 2-8oC. For maximal stability, apportion the reconstituted preparation into working aliquots and store at -20oC to -70oC. Avoid repeated freeze/thaw cycles.