Affinity purification using immunoprecipitation (IP) is an extremely useful method for target profiling of bioactive natural products. We examined IP purification of CMetE, which is a molecular target for potassium isolespedezate (1), a leaf-opening factor of Cassia plant. We studied IP efficiency using a panel of FLAG-connected molecular probes (2-8), including probes with varying structures and lengths of the linker moiety. The results suggest that not only the length, but the chemical nature of the linker moiety, strongly affect the IP efficiency. 3XFLAG, a tag combined with a linker moiety of charged amino acids, gave the best results and was most useful for IP purification of the molecular target.
- Molecular probe
- Target protein