Dear Shijun, Just a reminder that the pI calculated from the sequence is not necessarily the real pI, and sometimes can very quite a bit due to folding and oligomerization of the protein. The first protein I ever expressed had a calculated pI of 9.2 and a real pI of 7.8 (if I remember correctly), so the difference can be quite large. You can determine the real pI by iso-electric focussing. Best wishes, Mark
Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC calle Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 Section Editor Acta Crystallographica F https://journals.iucr.org/f/ > On 20 Jul 2020, at 08:38, 张士军 <[email protected]> wrote: > > Dear: > > The protein was purified in 4 degree, and the expression level is low, so the > aggregation is not by high concentration; the buffer pH is 7.5 which is not > colse to the PI 8.6. It should be a dimer when function, but it was > aggregated when negative staining. Maybe I could try to add arginine when > purification, or do mutantions. anyone has website for prediction the > mutation sites of protein? > > Thanks! > > Best, > > shijun > > > > -----原始邮件----- > 发件人:"Nikolay Dobrev" <[email protected]> > 发送时间:2020-07-19 20:29:49 (星期日) > 收件人: [email protected] > 抄送: > 主题: Re: [ccp4bb] protein oligomer > > It really depends from the nature of the protein and if is > oligomerizing/agregating/forming polymers if any of this is reversible. > On the other side if you are working with one of the fibril forming protein > it will require optimizaiton on its on as they will form naturally polymers. > > Do you observed different specises when you analyze your protein by SEC or if > you are able to perfom DLS? > Additional information regarding your protein will be really helpful for more > detalied suggestions how to overcome your protein. > > Best, > Nikolay > > Nikolay Dobrev > Scientific Officer, Protein Expression and Purification Core Facility > EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany > T +49 6221 387 8633 | M +49 173 684 0532 > twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia > Visit www.embl.org/events <http://www.embl.org/events> for a complete list of > all EMBL events. > > On 19/07/2020 14:15, S. Mohanty wrote: >> Keep the protein concentration low during purification steps along with >> using other anti-aggregation agent/s. Make sure that the pH at which you are >> purifying is not close to the pI of the protein. Until completely purified, >> all purification steps should be performed in a cold room if it is a soluble >> protein. >> >> Smita >> >> >> On Sunday, July 19, 2020, 04:28:45 AM CDT, Sorin Draga <[email protected]> >> <mailto:[email protected]> wrote: >> >> >> I am not sure what you mean by polymer formation. Presuming that you have >> optimized your protein concentration, pH and salt concentration, you could >> try arginine as an anti-aggregation agent in your purification (I presume >> you do FPLC). Have a look at chaotropic agents used in protein purification, >> The answer is generally dependent on the protein/proteins you are trying to >> purify and is not necessarily straightforward. >> >> Kinds regards >> >> On Sun, Jul 19, 2020 at 12:08 PM 张士军 <[email protected] >> <mailto:[email protected]>> wrote: >> Dear all: >> >> Any ideas to decrease protein polymer formation? my protein was easy to form >> oligomers and precipitation when do purification,I have tried add glycerol >> and DTT,both didn't work. Does anyone has experience to avoid it happening. >> Thanks! >> >> Best Regards >> >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1>-- > Nikolay Dobrev > Scientific Officer, Protein Expression and Purification Core Facility > EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany > T +49 6221 387 8633 | M +49 173 684 0532 > twitter.com/EMBLorg | facebook.com/embl.org | 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&A=1 > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=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/
