Re: [ccp4bb] finicky protein
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 [EMAIL PROTECTED] 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 + From: Mads Gabrielsen <[EMAIL PROTECTED]> 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 GlasgowPhone Office: 01413308119 G12 8QQ Phone Lab: 01413306449 UK E-mail: [EMAIL PROTECTED] 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
Re: [ccp4bb] finicky protein / cell disruption
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 press (or a cell emulsifier), disruption at low pressures (e.g. three passes at low pressure) increases the level integrity of full complexes, while giving good yield. My recommendation is to throw your sonicator away and use methods that apply less heat and energy to your cells and to your biomolecules. - Th Thomas Earnest, Ph.D. Senior Scientist and Group Leader Structural Proteomics Development Group Physical Biosciences Division MS64R0121 Lawrence Berkeley National Laboratory Berkeley CA 94720 [EMAIL PROTECTED] 510 486 4603 Mads Gabrielsen wrote: 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
Re: [ccp4bb] finicky protein
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 + >From: Mads Gabrielsen <[EMAIL PROTECTED]> >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 GlasgowPhone Office: 01413308119 >G12 8QQ Phone Lab: 01413306449 >UK E-mail: [EMAIL PROTECTED] > > >This message was sent using IMP, the Internet Messaging Program.
Re: [ccp4bb] finicky protein
Quoting James Stroud <[EMAIL PROTECTED]>: Try refolding before purification. OR, you could do a denaturing purification, followed by refolding. Ni-NTA's perfectly viable in denaturing urea, for example. From there, it's just a question of whether to try refolding on-resin, or following elution. Cheers, d.s. === David M. Shechner, Graduate Student Bartel Laboratory Department of Biology Whitehead Institute for Biomedical Research Massachusetts Institute of Technology [EMAIL PROTECTED] ===
Re: [ccp4bb] finicky protein
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 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 -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com
Re: [ccp4bb] finicky protein
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 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 -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com
Re: [ccp4bb] finicky protein
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 restricted proteolysis - well, you need a little bit of purified protein for this, but it might be the most promising - guesstimate a proper subclone from secondary structure prediction of your protein. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Sun, 2 Mar 2008, Naren sharma 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
