Hi Umar Artem is right, we should have some more infos about how you proceed after disrupting the cells. The part I find curious is your usage of "usually". Usually soluble when? Before you centrifugate the sample? Before you go into the äkta? That would be a first hint to know what you mean by "usually"..
best, matthias Dr. Matthias Barone AG Kuehne, Rational Drug Design Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Strasse 10 13125 Berlin Germany Phone: +49 (0)30 94793-284 ________________________________ From: CCP4 bulletin board <[email protected]> on behalf of Umar Farook <[email protected]> Sent: Saturday, June 27, 2020 6:08:51 PM To: [email protected] Subject: Re: [ccp4bb] Looking for suggestions with protein expression Glutathione beads for GST tagged protein. Umar On Sat, 27 Jun 2020, 6:56 pm John Newitt, <[email protected]<mailto:[email protected]>> wrote: On Jun 27, 2020, at 3:15 AM, Umar Farook <[email protected]<mailto:[email protected]>> wrote: > > > Dear All, > > Sorry for an offtopic question, your suggestions are highly appreciated. > > We have been working on iron sulfur cluster binding protein, which is usually > expressed as a nice soluble protein expressed in BL21 cells but aggregated in > the affinity column itself and unable to recover from it. We had made n > number of truncations and fused to soluble tags such as MBP, but always ended > up in large aggregates. Anyone has experience in working with iron-sulfur > cluster binding protein before, please let us know the critical steps in > purification of such proteins, whether you have completely done the > expression, purification and crystallization in anaerobic conditions? or else > changing the expression system to eukaryotic system such as Baculo or HEK > 293T would help? > > Please share your valuable experience, thank you. > > When you say “affinity column”, are you referring to a Ni2+-affinity column? -John ________________________________ 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/
