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

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


protein purification!

protein purification! - Protocols and Methods Forum

protein purification! - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 06-15-2007, 05:02 PM
Li-Hua Zhu
Guest
 
Posts: n/a
Default protein purification!



Dear all,

We are now trying to purify a recombinant protein (his-tag) produced
in E. coli. This protein (inclusion body) is only soluble in high
salt with low pH (4,5). (we usually dissolve it in 8M guanidine at pH
4,5). The protein was purified using his-tag column, but there are
still two extra bands after column purification. We have produced
antiserum in rabbits using the column-purified protein and the
antiserum is going to be purified by an affirnity column, which will
be performed in a commercial company. For this purpose, we need to
produce pure protein so that the commany can use it as a kind of
positive control. We want to elute the protein band from a SDS-PAGE
gel. It has been difficult due to the insolubility of the protein in
most solutions. We tried a commercial available kit to dissolve the
gel, but the gel was not completely dissolved and no protein was
found in the supernatant. Do you have any experience in dissolving
the gel while maintaining the protein's structure unchanged? Any help
is highly appreciated.


Lihua

Reply With Quote
  #2  
Old 06-15-2007, 10:21 PM
DK
Guest
 
Posts: n/a
Default protein purification!

In article <[Only registered users see links. ].net> , Li-Hua Zhu <[Only registered users see links. ].se> wrote:

What exactly is the affinity column going to be??? If the protein
is misfolded/denatured, the value of such an affinity step is
questionable at best - antibodies against short epitopes will be
selected but also a lot of non-specific garbage will be "purified"
owing to hydrophobic interactions.


You will be better off trying to find conditions that produce partial
refolding of your protein and purifying it. This renatured stuff should be
coupled to the insoluble support. There is tons of literature on the subject
and every other company sells a refolding kit that is a lot better than
other refoldng kits :-)

DK

Reply With Quote
Reply

Tags
protein , purification


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 03:06 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.16778 seconds with 16 queries