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" <nikolay.dob...@embl.de> 发送时间:2020-07-19 20:29:49 (星期日) 收件人: CCP4BB@JISCMAIL.AC.UK 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 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 <sor.dr...@gmail.com> 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 <21620150150...@stu.xmu.edu.cn> 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 To unsubscribe from the CCP4BB list, click the following link: 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 -- 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 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 ######################################################################## 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/
