ab4754 was tested by immunoblot against Saccharomyces cerevisiae lysates expressing the Hub1-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 1/1000 dilution of Goat polyclonal to GFP (ab6673) followed by reaction with a 1/15000 dilution of Donkey polyclonal to Goat IgG (HRP)(ab7125).  Panel C shows specific reaction with Hub1 using a 1/500 dilution of ab4754 followed by reaction with a 1/15000 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 S