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


weak binding to ni-nta column under denaturing conditions

weak binding to ni-nta column under denaturing conditions - Protein Forum

weak binding to ni-nta column under denaturing conditions - Protein Forum


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 07-21-2005, 09:09 AM
lea
Guest
 
Posts: n/a
Default weak binding to ni-nta column under denaturing conditions



I am expressing a protein with c-terminal his-tag in bl21(de3) cells as
inclusion bodies. My problem is that even under denaturating conditions
binding of the protein to the ni-nta qiagene column is very low. In
fact, no binding occures if i follow a denaturating protocol (changing
buffer pH values). Some of the protein binds to the column if native
imidazole (10, 20, 250) protocol supplemented with 8M urea is followed,
but still majority of protein is eluted in wash fraction. To avoid the
column flow problem i did several mini-preps with prolonged incubations
and stirring options but the result remains the same. Increasing the
total salt concentration up to 2M did not help, neither did addition of
2-mercaptoethanol. I cant afford anti-his antibodies and will try
adding more protease inhibitors. Any suggestions or experience with
changing the his-tag orientation or maybe even adding a second his-tag?
Thanks,

Lea

Reply With Quote
Reply

Tags
binding , column , conditions , denaturing , ninta , weak


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
protein purification magnificientleo Proteomics Forum 11 12-29-2010 09:01 AM
His-tag protein purification under denaturing conditions Arabidop Molecular Biology Articles and Protocols 5 06-28-2009 06:05 AM
Weak binding of His-tags to IMAC Bean Long Protocols and Methods Forum 9 09-23-2005 05:57 AM
Sequence non-specific DNA binding EK Protocols and Methods Forum 5 08-28-2003 02:41 AM


All times are GMT. The time now is 09:52 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 0.10958 seconds with 16 queries