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Hey all,
I did a western the other week and used two primary antibodies (mouse anti-actin and rabbit anti-IL7R) and then used two secondaries, (goat anti-mouse, and goat anti-rabbit). Both secondaries are labelled with IR dyes, the anti mouse is 680nm, the anti-rabbit is 800nm.
I admit, I transferred the proteins to PVDF, then incubated with both primaries together, and then both secondaries together. I ended up with a lovely yellow band at the size of actin, and nothing at the size of IL-7R. Have I screwed it up? Should I incubate everything separately? Which order would be best? I figured since I had different species and targets I would be ok to incubate it all together....
