Hello, I am able to enrich and immunoprecipitate a very large glycoprotein from human epithelial cells. I suspect that it undergoes ectodomain shedding as I can detect it in the media. To analyze trafficking I attempted to use S35 labeling which repeatedly failed with only background bands showing up on the radiogram. Various pre-clearing steps to reduce background did not help. Is it possible that S35 pulse chase is simply not suitable to study proteins expressed at very low levels? Does Cycloheximide chase substitute for S35 pulse chase?
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