HomeFAQsI see my intact fusion protein by SDS-PAGE when I run cells boiled in Sample Buffer, but when I check the crude extract the fusion is degraded.
FAQ: I see my intact fusion protein by SDS-PAGE when I run cells boiled in Sample Buffer, but when I check the crude extract the fusion is degraded.
For fusions expressed in the cytoplasm, in many cases most of the degradation happens during harvest and lysis. Harvesting promptly and lysing the cells quickly may help. In other cases, degradation occurs when the fusion protein is exposed to periplasmic or outer membrane proteases. The best strategy in either case is to use a host which is deficient in the offending protease(s).
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