Hi to everyone!
I have to validate endotoxins removal from DEAE-Sepharose I use for the purification of my hormone. The resin undergoes to a sanitization protocol with NaOH. To demonstrate the efficiency of NaOH to inactivate endotoxins I spike them into the resin, pack the resin in a column, run a sanitization and then I determine the endotoxins amount in the resin before and after the sanitization. My problem is that when I spike the DEAE-Sepharose with endotoxins, I can't quantified them anymore in the slurry (LAL kinetic chromogenic test), probably because endotoxins charged negatively bind the functional groups DEAE (diethylaminoethyl) that are charged positively!!! In this way I can't define logarthimic reduction because I can't determine the initial concentration of endotoxins in the resin since I found 0!
Someone could help me to solve this problem? I tried using salt or changing pH buffers to remove endotoxins from the resin but with no success!!! Thanks a lot