Vector control, Miro1-HA and Miro2-HA were transiently expressed in HEK 293 cells and whole cell lysates were made. 25ug of lysates were used to confirm specificity of the Miro2 antibody. 1ug/ml of anti-Miro2 antibody was used for immunoblotting. We expressed Miro1, an orthologue of Miro2 to see if the antibody can cross-react with Miro1. The antibody specifically recognized over-expressed Miro2 and also recognized endogenous Miro2. Both endogenous and over-expressed protein ran at 75 kDa and we realized that Miro-HA over-expression is only twice as much as endogenous as we observe a slight increase in band intensity in the Miro2 over-expressed lane.
100ug of whole cell lysates from HEK293 cells was immunoprecipitated for endogenous Miro2 either using 5ug anti-Miro2 antibody covalently coupled to Protein-G sepharose beads or with 5ug pre-immune sheep IgG coupled to protein- G sepharose. 25ug of HEK 293 whole cell lysate was ran as an input control for the immunoprecipitation. We observe that the Miro2 antibody can specifically pull-down endogenous Miro2 as we do not observe any band in the IgG control and we see a band of the correct molecular weight when the pull down was performed with Miro2 antibody.