Anti-Nedd8 antibody (ab4751)

  Western blot - Nedd8 antibody (ab4751)

ab4751 tested by immunoblot against a yeast cell lysate. A dilution of the antibody between 1:200 and 1:1,000 will show strong reactivity specifically with free Rub1 protein (indicated by arrow) and Rub1 conjugates. In this blot the antibody was used at a 1:500 dilution incubated overnight at 4° C in 5% non-fat dry milk in TTBS. Detection occurred using a 1:2000 dilution of Donkey polyclonal to Rabbit IgG (HRP) (ab7083) for 1 hour at room temperature. A chemiluminescence system was used for signal detection (Roche).

  Western blot - Nedd8 antibody (ab4751)

Immunoblot of Rub1 fusion protein. Anti-Rub1 antibody generated by immunization with recombinant yeast Rub1 was tested by immunoblot against yeast lysates expressing the Rub1-GFP fusion protein and other UBL fusion proteins. All UBLs possess limited homology to Ubiquitin and to each other, therefore it is important to know the degree of reactivity of each antibody against each UBL. Panel A shows total protein staining using ponceau. Panel B shows positions of free GFP or GFP containing recombinant proteins present in each lysate preparation after reaction with a 1:1,000 dilution of Goat polyclonal to GFP (ab6673) followed by reaction with a 1:15,000 dilution of Donkey polyclonal to Goat IgG (ab7125). Panel C shows specific reaction with Rub1 using a 1:1,000 dilution of ab4751 followed by reaction with a 1:15,000 dilution of Goat polyclonal to Rabbit IgG (HRP) (ab7090). All primary antibodies were diluted in TTBS buffer supplemented with 5% non-fat milk and incubated with the membranes overnight at 4° C. Yeast lysate proteins were separated by SDS-PAGE using a 15% gel. This data indicates that anti-Rub1 is highly specific and does not cross react with other UBLs, however, in other experiments (not shown) some cross reactivity is observed against ubiquitin. Include an anti-ubiquitin control any experiment where the specific detection of Rub1 is uncertain. Note that two bands are detected by anti-GFP indicating that most Rub1-GFP is processed in yeast. A chemiluminescence system was used for signal detection (Roche). Data contributed by M. Malakhov, www.lifesensors.com, personal communication.