Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > Protein Forum
Register Search Today's Posts Mark Forums Read

Protein Forum Protein Forum


Is there a way to enhance yield of soluble DNA-binding proteinin limiting DNA-protein interactions ?

Is there a way to enhance yield of soluble DNA-binding proteinin limiting DNA-protein interactions ? - Protein Forum

Is there a way to enhance yield of soluble DNA-binding proteinin limiting DNA-protein interactions ? - Protein Forum


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 02-04-2008, 03:39 PM
Nicolas BAZEILLE
Guest
 
Posts: n/a
Default Is there a way to enhance yield of soluble DNA-binding proteinin limiting DNA-protein interactions ?



hi,


I try to purify a protein from yeast to assay some structural studies.
unfortunatly, the yield is really poor and I want to find some ways to
increase it. First, I change the medium and the temperature and the time of
induction but I think that now it is optimal.
Because my protein is a DNA binding protein, I think that I lost some
proteins fixed on genomic DNA that are found in the pellet during the step
of clarification just after breaking cells. Does it may be right ? Is there
a way to prevent this interactions (high salt concentration, additives ??)
I ask also, to know if there is a special manipulation to get a clear
pellet during the step of cells collecting because my pellet is rapidly
resolublize and the end of the run in spite of an optimal speed, resulting
in the loss of some cells. Is there specials additives to get clear,
compact and well packed pellet of cells, that I can put directly on the
medium (YNB -URA galactose) at the end of induction.


Nicolas pHD student Institut Curie Paris france


Reply With Quote
  #2  
Old 02-20-2008, 02:28 PM
incrediblyhigh@hotmail.com
Guest
 
Posts: n/a
Default Is there a way to enhance yield of soluble DNA-binding protein inlimiting DNA-protein interactions ?

On Feb 4, 3:39*pm, Nicolas BAZEILLE <[Only registered users see links. ]>
wrote:

Why not add some DNase to you lysis solution?? That should get rid of
most DNA. Not having worked with yeast, can you sonicate the cells?
That should also help in shearing the DNA.

Can't help you with the cell pelleting problem I'm afraid...

I agree with clement in that you should first look at total expression
but maybe you already have: is the yield poor after purification or do
you simply have low levels of expression?


Assuming the problem lies with purification:
High salt can help... in fact, the low yield may also be a consequence
of not using the right buffer conditions for your protein. Ideally
you would do some small scale lysis/purification experiments to
determine the optimum pH and concentration of salt for your protein
during lysis and subsequent steps.
Does it have cysteines? If so, adding reducing agents might help...
Low quantities of detergent (eg triton x100 or tween20, 0.01% or less)
can help as well...


Reply With Quote
Reply

Tags
dnabinding , dnaprotein , enhance , interactions , limiting , proteinin , soluble , yield


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
Human Cytome Project - an idea - Update 19 April 2005 Peter Van Osta Cell Biology and Cell Culture 1 06-01-2009 02:17 PM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM
New Saccharomyces Sequences 11/27/04 Mike Cherry Yeast Forum 0 11-29-2004 12:39 AM
New Saccharomyces Sequences 05/19/04 SGD Sequences Yeast Forum 0 05-23-2004 04:06 PM


All times are GMT. The time now is 03:57 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.17127 seconds with 16 queries