Re: [ccp4bb] finicky protein

2008-03-05 Thread Juergen Bosch
chunks, which can change how DNA binding proteins behave in the lysate. Original message Date: Mon, 3 Mar 2008 15:21:15 + From: Mads Gabrielsen [EMAIL PROTECTED] Subject: [ccp4bb] finicky protein To: CCP4BB@JISCMAIL.AC.UK I am not a big fan of sonication. Try changing

Re: [ccp4bb] finicky protein

2008-03-03 Thread James Stroud
Try refolding before purification. On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote: Hi all sorry, for offtopic query... I am trying to purify my protein by Ni-NTA affinity chromatography. After sonication as i centrifuge bacterial lysate, soon after 10 min whole lysates get precipitated

Re: [ccp4bb] finicky protein

2008-03-03 Thread Brenda Patterson
Did you filter your lysate through .45 then .22 filters? cheers b Quoting James Stroud [EMAIL PROTECTED]: Try refolding before purification. On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote: Hi all sorry, for offtopic query... I am trying to purify my protein by Ni-NTA affinity

Re: [ccp4bb] finicky protein / cell disruption

2008-03-03 Thread Thomas Earnest
I will second this recommendation to use alternate cell disruption methods instead of sonication. Particularly with multi-protein complexes, where the preservation of the complex from disruption though crystallization is important, sonication can be quite deleterious. Even with the French

[ccp4bb] finicky protein

2008-03-02 Thread Naren sharma
Hi all sorry, for offtopic query... I am trying to purify my protein by Ni-NTA affinity chromatography. After sonication as i centrifuge bacterial lysate, soon after 10 min whole lysates get precipitated during loading on the column and some time it remain soluble too. if i get purified through

Re: [ccp4bb] finicky protein

2008-03-02 Thread Tim Gruene
If you really tried all sorts of buffers (did you go to extreme pH-values, very high salt concentrations, tried various additives (beta-ME, glycerol,...), different salts) you can still - try another expression system (insect cells, mammalian) - see if any modifications might be useful - try