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

Molecular Biology Techniques Molecular Biology Forum. Includes forums for common molecular biology techniques.


Low protein yield

Low protein yield - Molecular Biology Techniques

Low protein yield - Molecular Biology Forum. Includes forums for common molecular biology techniques.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 03-29-2012, 04:32 PM
Pipette Filler
Points: 22, Level: 1 Points: 22, Level: 1 Points: 22, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2012
Location: Miami
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Low protein yield



Hello!
I am trying to purify and refold my his-tagged protein on ni-nta column using urea gradient from 8M to 2M at room temperature. The buffer also has 0.25% Triton and 5mM BME. Then I wash with a buffer without urea, Triton, and BME and then elute with low pH (4.0). I am using gravity column, so it's quite slow and takes me several hours. I am consistently getting a very low yield (only around 1%). I checked the flow-through and the column for protein by SDS-PAGE, didn't see any bands. I think the protein might be degraded on the column, however, it is supposed to be pretty stable even at room temperature. Does anyone have any suggestions on what I could try to get better yield? Perhaps, I run the column for too long? I know that EDTA and PMSF inhibit proteases but I think they will also react with the column, so I can't use those. Did anyone try using, for example, pepstain or benzamidine with ni-nta?
Please help figure out the problem! Thanks.
Reply With Quote
Reply

Tags
low , protein , 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 03:01 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 0.16420 seconds with 16 queries