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
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
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
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
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
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
