Dear CCP4BB members, We would like to identify a ligand that is present in crystal structure (according to strong positive densities at active site) but absent in crystallization condition. We already have some candidates in mind based on our knowledges on this protein but we need to validate further. The general method we are using now is to use methanal to precipitate protein and extract ligand from the precipitated protein. Then we analyse the methanol extraction sample on LC-MS. One problem of this method is that the methanol extraction will not be 100% efficient which means there is only a small portion of bound-ligand can be extracted from the protein— particularly if the ligand binds very tightly to the protein. So I would like to know whether anyone has experience to efficiently extract tighly-bound ligands from protein for downstream analysis. One method is to digest protein with protease such as trypsin. Or use urea to denature the protein. However, these methods require relatively long processing time which is not optimal when the ligand that we want to analyse is unstable (degrade overtime). Anyone has more suggestions?
Thank you! Kind regards, Wenhe
