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). 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 r

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 mi