The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml. Detects a band of approximately 24 kDa (predicted molecular weight: 21 kDa).
Use a concentration of 1 µg/ml.
Accepts ubiquitin from the E1 complex and catalyzes its covalent attachment to other proteins. Catalyzes the covalent attachment of ISG15 to other proteins. Mediates the selective degradation of short-lived and abnormal proteins. In vitro also catalyzes 'Lys-48'-linked polyubiquitination.
Protein modification; protein ubiquitination.
Belongs to the ubiquitin-conjugating enzyme family.
UBCH6/UBE2E1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to UBCH6/UBE2E1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab36980. Secondary: Clean blot (HRP conjugate) at 1/1000 dilution. Band: 25kDa: hnRNP A1; non specific - 18kDa: We are unsure as to the identity of this extra band.
ICC/IF image of ab36980 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab36980, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
Hans F et al. UBE2E ubiquitin-conjugating enzymes and ubiquitin isopeptidase Y regulate TDP-43 protein ubiquitination. J Biol Chem289:19164-79 (2014).
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