Re: [ccp4bb] Auto-proteolysis

[Boaz Shaanan]

Hi, How certain are you that what you see is the result of auto-proteolysis and not proteolysis  by a contaminating protease? Have you used protease inhibitors (of all sorts) during purification?            Boaz     Boaz Shaanan, Ph.D.                                         Dept. of Life Sciences                                      Ben-Gurion University of the Negev                          Beer-Sheva 84105                                            Israel                                                                                                                  E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220  Skype: boaz.shaanan                  Fax:   972-8-647-2992 or 972-8-646-1710                          From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Mohammad Khan [mohdkhan0...@gmail.com] Sent: Tuesday, May 26, 2015 8:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Auto-proteolysis Dear all, I am working on a His-tag recombinant protein with two domains, which I purify using affinity chromatography. When I set up crystallization of the same, it gave me crystals in two different conditions- one was the complete protein. The other just had the Domain2. Even on SDS-PAGE, I see three different bands of the purified protein. How can I determine the autu-proteolysis of this protein? This class of protein has not reported to have any auto-proteolytic properties, though it forms isopeptide bonds by autocatalysis. Thanks a lot Mohammad