Re: [ccp4bb] finicky protein

Protein crystallography Programs for crystallography X-ray detectors
Basic tutorials:
Protein Peptide

CCP4bb navigation

CCP4bb <-- 2008 <-- March 2008 <-- 03 March 2008

Previous message:
Subject: finicky protein
From: Mads Gabrielsen mg118m {- at -} UDCF {- dot -} GLA {- dot -} AC {- dot -} UK
Date: 2008-03-03

Next message:
Subject: Re: finicky protein / cell disruption
From: Thomas Earnest TNEarnest {- at -} LBL {- dot -} GOV
Date: 2008-03-03

Subject: Re: finicky protein
From: price {- at -} UCHICAGO {- dot -} EDU price {- at -} UCHICAGO {- dot -} EDU
Date: 2008-03-03

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
>Subject: [ccp4bb] finicky protein
>To: CCP4BB@JISCMAIL.AC.UK
>
>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:
m.gabrielsen@bio.gla.ac.uk
>
>------------------------------------------------------------
----
>This message was sent using IMP, the Internet Messaging
Program.

CCP4bb navigation

CCP4bb <-- 2008 <-- March 2008 <-- 03 March 2008

Previous message:
Subject: finicky protein
From: Mads Gabrielsen mg118m {- at -} UDCF {- dot -} GLA {- dot -} AC {- dot -} UK
Date: 2008-03-03

Next message:
Subject: Re: finicky protein / cell disruption
From: Thomas Earnest TNEarnest {- at -} LBL {- dot -} GOV
Date: 2008-03-03