Dear Wenhe, we had a similar case and extraction using CHCl3 plus CH3OH (2:1 ratio, v/v) (65 °C, 30 min) before mass spectrometry worked very well: https://www.ncbi.nlm.nih.gov/pubmed/21743455 Hope that helps, Tomas
On Mon, Aug 21, 2017 at 4:41 AM, WENHE ZHONG <wenhezhong.xmu....@gmail.com> wrote: > 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
