Re: [ccp4bb] [External] Re: [ccp4bb] protease inhibitor and 3c protease
Dear Dhiraj, yes, on-column cleavage works as well quite efficiently with 3C protease. I am performing it in a cold room (8-10C) for 1h normally reaches similar efficiency. Depending on your tags (if the 3C has a His-tag) you can add even more protease 1:50 then for sure 1h is more than enough. The only thing you need to ensure is that upon cleavage your protein does not bind to the IMAC resin due to internal Histidines. If that is the case you can, just collect the flow-through and pass it over the beads couple of more times to ensure efficient binding of the His-3C protease then you should have a highly pure target. Please note, I am referring to Ni-NTA Agarose beads, which you use in bach mode. The efficiency will be lower most like if you are using pre-packed columns. I hope that is helpfull. Best, Nikolay > On 02/03/2022 6:56 PM Srivastava, Dhiraj > wrote: > > > Thanks Nikolay > I am doing on column cleavage, so I am > incubating it overnight at 4-degree C. Do you think even on column digestion > is also going to be done in one hour. > > Thank you > Dhiraj > > > - > From: CCP4 bulletin board on behalf of Nikolay > Dobrev > Sent: Thursday, February 3, 2022 11:53 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [External] Re: [ccp4bb] protease inhibitor and 3c protease > > Dear Dhiraj, > the 3C protease is from the Cysteine protease family. > Therefore for sure, you can add EDTA (if it is not indented for follow up > IMAC purification) and that will inhibit most of the metalloprotease. > Furthermore, you can resort to Serine type protease inhibitors like PMSF or > Aprotinin. > > One side not, the 3C cleavage does not need to be long, as it is an > extremely potent protease. If the cleavage sequence is well accessible in > less than 1 hour at 4*C you can obtain >95% cleavage when you use 1:100 ratio > protease to substrate. Increasing the temperature speeds up the cleavage > dramatically and at room temperature, you can obtain the same efficiency for > 10 mins approx. > I am assuming you are interested in using the 3C protease to cleave a tag > from recombinantly expressed protein. > > > Kind regards, > Nikolay > > > > > On 02/03/2022 6:25 PM Srivastava, Dhiraj > wrote: > > > > > > Hi > > sorry for off topic question. does anyone know which protease > > inhibitors we can include safely while cleaving with 3C protease? > > > > Thank you > > Dhiraj > > > > > > - > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > > > > Nikolay Dobrev > Postdoctoral Fellow @ Wilmanns group > EMBL Hamburg, c/o DESY, Building 25A, > Notkestraße 85, 22607 Hamburg, Germany > T +49 40 89902 165 | M +49 173 684 0532 > twitter.com/emblevents https://twitter.com/emblevents > |http://facebook.com/embl.org | http://youtube.com/user/emblmedia > Visit http://www.embl.org/events for a complete list of all EMBL events. > > > > - > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > > - > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > Nikolay Dobrev Postdoctoral Fellow @ Wilmanns group EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22607 Hamburg, Germany T +49 40 89902 165 | M +49 173 684 0532 twitter.com/emblevents https://twitter.com/emblevents |http://facebook.com/embl.org | http://youtube.com/user/emblmedia Visit http://www.embl.org/events for a complete list of all EMBL events. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] [External] Re: [ccp4bb] protease inhibitor and 3c protease
Thanks Nikolay I am doing on column cleavage, so I am incubating it overnight at 4-degree C. Do you think even on column digestion is also going to be done in one hour. Thank you Dhiraj From: CCP4 bulletin board on behalf of Nikolay Dobrev Sent: Thursday, February 3, 2022 11:53 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [External] Re: [ccp4bb] protease inhibitor and 3c protease Dear Dhiraj, the 3C protease is from the Cysteine protease family. Therefore for sure, you can add EDTA (if it is not indented for follow up IMAC purification) and that will inhibit most of the metalloprotease. Furthermore, you can resort to Serine type protease inhibitors like PMSF or Aprotinin. One side not, the 3C cleavage does not need to be long, as it is an extremely potent protease. If the cleavage sequence is well accessible in less than 1 hour at 4*C you can obtain >95% cleavage when you use 1:100 ratio protease to substrate. Increasing the temperature speeds up the cleavage dramatically and at room temperature, you can obtain the same efficiency for 10 mins approx. I am assuming you are interested in using the 3C protease to cleave a tag from recombinantly expressed protein. Kind regards, Nikolay On 02/03/2022 6:25 PM Srivastava, Dhiraj wrote: Hi sorry for off topic question. does anyone know which protease inhibitors we can include safely while cleaving with 3C protease? Thank you Dhiraj To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 Nikolay Dobrev Postdoctoral Fellow @ Wilmanns group EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22607 Hamburg, Germany T +49 40 89902 165 | M +49 173 684 0532 twitter.com/emblevents<https://twitter.com/emblevents> | facebook.com/embl.org<http://facebook.com/embl.org> | youtube.com/user/emblmedia<http://youtube.com/user/emblmedia> Visit www.embl.org/events<http://www.embl.org/events> for a complete list of all EMBL events. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] protease inhibitor and 3c protease
Dear Dhiraj, the 3C protease is from the Cysteine protease family. Therefore for sure, you can add EDTA (if it is not indented for follow up IMAC purification) and that will inhibit most of the metalloprotease. Furthermore, you can resort to Serine type protease inhibitors like PMSF or Aprotinin. One side not, the 3C cleavage does not need to be long, as it is an extremely potent protease. If the cleavage sequence is well accessible in less than 1 hour at 4*C you can obtain >95% cleavage when you use 1:100 ratio protease to substrate. Increasing the temperature speeds up the cleavage dramatically and at room temperature, you can obtain the same efficiency for 10 mins approx. I am assuming you are interested in using the 3C protease to cleave a tag from recombinantly expressed protein. Kind regards, Nikolay > On 02/03/2022 6:25 PM Srivastava, Dhiraj > wrote: > > > Hi > sorry for off topic question. does anyone know which protease > inhibitors we can include safely while cleaving with 3C protease? > > Thank you > Dhiraj > > > - > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > Nikolay Dobrev Postdoctoral Fellow @ Wilmanns group EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22607 Hamburg, Germany T +49 40 89902 165 | M +49 173 684 0532 twitter.com/emblevents https://twitter.com/emblevents |http://facebook.com/embl.org | http://youtube.com/user/emblmedia Visit http://www.embl.org/events for a complete list of all EMBL events. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] protease inhibitor and 3c protease
Hi sorry for off topic question. does anyone know which protease inhibitors we can include safely while cleaving with 3C protease? Thank you Dhiraj To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] protease inhibitor
Hi All, Does any one add protease inhibitor to purified protein just before setting trays? If yes what is the typical % that should be added ? regards rashmi
[ccp4bb] Protease inhibitor cocktails and protein crystallization.
Dear All, I apologize if the questions has already been asked on this forum. We are purifying a membrane that seems prone to proteolysis. Although we use Protease Inhibitor cocktails during lysis and the first step of purification we get rid of them after and only keep PMSF and EDTA as general anti-protease control agents. I am considering reincluding the cocktail of inhibitors at the last purification step (a size exclusion in our case) and was wondering if having this infamous mixture of peptidic inhibitors (for the most part) around during crystallization would be a problem: specifically getting crystals of these inhibitors. Does anyone have extended experience in this matter. Many thanks in advance. -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Protease inhibitor cocktails and protein crystallization
Regarding adding inhibitors. I'm not sure the effect on crystallization but if you use too much you will get covalent modification of your protein from one of them (I can check my notes as to which one). We searched many MS databases of protein modifications to realize why our protein was the wrong Mr. Then my student happened to mention that he had added 10 times top much. Well at least we now know what not to do. Mark Saper sa...@umich.edu
