Something else you could try is adding know ligands to your purification
step - depending on your proteein (or even during expression, e.g.
metals come to mind).
Juergen
[log in to unmask] wrote:
>Just beware that changing how you break the cells open can
>change the average size of chromosome chunks, which can
>change how DNA binding proteins behave in the lysate.
>
>
>---- Original message ----
>
>
>>Date: Mon, 3 Mar 2008 15:21:15 +0000
>>From: Mads Gabrielsen <[log in to unmask]>
>>Subject: [ccp4bb] finicky protein
>>To: [log in to unmask]
>>
>>I am not a big fan of sonication. Try changing your way of
>>
>>
>disrupting the
>
>
>>cells.
>>
>>I have compared sonication vs mechanical stress on several
>>
>>
>unrelated proteins,
>
>
>>and for me a good old french press wins every time. If you
>>
>>
>want to get all
>
>
>>modern and fancy, a cell disruptor gives similar results.
>>
>>Cheers,
>>
>>Mads Gabrielsen
>>
>>
>>[Hide Quoted Text]
>>
>>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 during loading on the column and some time
>>
>>
>it remain
>
>
>>soluble too. if i get purified through the column without
>>
>>
>precipitation,
>
>
>>it
>>gets precipitated during dialysis.
>>I have tried lot, by chnaging buffers, increasing salt or
>>
>>
>deacreasing salt
>
>
>>or no salt at are helpless.
>>I do purifiaction in cold room.
>>
>>can any one suggest some solution?
>>
>>Thanks in advance.
>>
>>NSH
>>
>>
>>--
>>Dr. Mads Gabrielsen
>>
>>GBRC, B217
>>Division of Biochemistry and Molecular Biology
>>IBLS
>>University of Glasgow Phone Office: 01413308119
>>G12 8QQ Phone Lab: 01413306449
>>UK E-mail:
>>
>>
>[log in to unmask]
>
>
>>------------------------------------------------------------
>>
>>
>----
>
>
>>This message was sent using IMP, the Internet Messaging
>>
>>
>Program.
>
>
>
--
Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
1705 NE Pacific Street
Seattle, WA 98195
Box 357742
Phone: +1-206-616-4510
FAX: +1-206-685-7002
Web: http://faculty.washington.edu/jbosch
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