I would try it using a lower blocking conditions than the Odyssey buffer for all your incubations - e.g., BSA and/or milk 1-5%. Then use HRP-linked secondary with ECL to visualize. Some proteins are hard to see in the Odyssey system, in my experience. The purified positive control suggests the antibody works but you might need to optimize for your samples. Is it possible your POI is not highly expressed in your cells? Maybe you could induce expression in vitro as another positive control. Also you could check for published citations and see how others may have prepared the lysates.
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