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

Molecular Biology Articles and Protocols Post Molecular Biology and science Protocols, Reviews, and Articles Forum. You can be the author!


His-tag protein purification under denaturing conditions

His-tag protein purification under denaturing conditions - Molecular Biology Articles and Protocols

His-tag protein purification under denaturing conditions - Post Molecular Biology and science Protocols, Reviews, and Articles Forum. You can be the author!


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 07-21-2006, 09:52 PM
Pipette Filler
Points: 2,033, Level: 28 Points: 2,033, Level: 28 Points: 2,033, Level: 28
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2006
Posts: 4
Thanks: 0
Thanked 1 Time in 1 Post
Post His-tag protein purification under denaturing conditions



This purification protocol was optimized for a poorly expressing protein in E. coli under denaturing conditions.


Material recommended: Gradient maker pump system (e.g. BioRad BioLogic system, Pharmacia gradiFrac or HPLC, etc.)

1) Prepare several litters of bacterail culture: Make a pre-culture over-night (for example 500 ml) without IPTG induction.

2) Inoculate the desired volume of fresh culture medium by a 1/10 dilution of the pre-culture. Incubate at 37°C for around 1 hour. Add 0.5 to 2 mM IPTG. Let grow for 4 to 8 hours.

3) Collect cells by centrifugation.

4) Ressuspend the cells in 8 M urea, 1M NaCl, 10% glycerol, pH 8 (sonication buffer). Sonicate around 8 to 10 min in ice with for example 6 sec impulses at 15 to 20 amplitude.

Note: For a weakly expressed protein a very concentrate lysate is the best choice, so whatever the culture volume, the lysate volume should not exceed around 30-35 ml.

5) Make the best clarification you can! Specially for concentrate lysates, to minimize the risks of column occlusion. For example, a 45 min and 11500 rpm centrifugation will do the job.

6) Harvest the clear supernatant and add 0.5% Triton X-100 (reduced if possible) and 5-10 mM Imidazol. remove bubbles by degasing.

Note 1: For a weakly expressed protein, non-specific interactions are a serious problem. Thus, a quite stringent condition should be used. The condition described here is far from the maximum allowed (but gives good results already), so some improvements can be obtained by increasing NaCl or glycerol for example.

Note 2: NaCl reduces ionic interactions, glycerol and Triton X-100 reduce hydrophobic interactions, Imidazol reduces weak and endogenous histidine interactions.

Purification step:

7) Use a 1 ml Ni column (HisTrap from Pharmacia for example). Equilibrate it with sonication buffer (8M urea, 1M NaCl, etc) at pH 8.

8) Pass the lysate throw the column. Save the Flow-throw. Wash the columns with around 30 ml of urea buffer pH 8 (sonication).

Note: Elution with pH drop is the best choice under denaturing conditions. It allows a subsequent purification if needed by simply raising pH to 8.

9) Begin elution by increasing elution buffer (sonication buffer adjusted to pH 4.95) proportion with gradual increase or isocratic step. A gradient is better for a first run. Contaminants will eluate first as pH falls.

Note: Elution buffer pH is highly crucial! adjust it carefully to 4.95. A small 0.5 pH diference inluence the elution time of the tagged protein.

In principle, a step to 90% elution buffer amount can be performed to eliminate most of contaminants. Let it flow for around 20 ml. Jump to next step at 95% for about 10 ml.

10) Elute by a 100% elution buffer step (pH 4.95) for around 10 ml.

Note: These values are indicative. In a first run, a gradient with holds should be performed to find adapted conditions.

11) Analyse fractions by a SDS-PAGE and Coomassie staining.

Reagents usage and compatibility with his-tag purification
Reagent

Max. conc. allowed

Usage
Phosphate buffer
Tris buffer, NOT recommended
NaCl 2 M prevents ionic interactions
Imidazol histidine competitor
Glycerol 30 % prevents hydrophobic interactions
Triton X-100, NP-40, CHAPS 2 % detergent, prevents hidrophobic interactions
Urea 8 M denaturing agent
Reply With Quote
The Following User Says Thank You to Arabidop For This Useful Post:
admin (06-10-2009)
  #2  
Old 10-24-2007, 10:04 AM
Pipette Filler
Points: 943, Level: 17 Points: 943, Level: 17 Points: 943, Level: 17
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2007
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: His-tag protein purification under denaturing conditions

Hi,

Great protocol, although I think the best choise for elution is using above 250mM Imidazole.
Reply With Quote
  #3  
Old 10-31-2007, 07:12 PM
Pipette Filler
Points: 1,288, Level: 20 Points: 1,288, Level: 20 Points: 1,288, Level: 20
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Posts: 10
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: His-tag protein purification under denaturing conditions

250 mM imidazole has been plenty in my experience - especially if you use a cobalt column instead of nickel. I switched to cobalt because I get better purity of my protein off the column.
Reply With Quote
  #4  
Old 11-27-2007, 11:00 PM
Pipette Filler
Points: 841, Level: 16 Points: 841, Level: 16 Points: 841, Level: 16
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2007
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: His-tag protein purification under denaturing conditions

Thank p topic beautiful
Reply With Quote
  #5  
Old 06-10-2009, 04:04 AM
Pipette Filler
Points: 2, Level: 1 Points: 2, Level: 1 Points: 2, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: His-tag protein purification under denaturing conditions

Hi,

Would you mind telling me where I can buy this kind of his-tag column?

Thank you very much.
Reply With Quote
  #6  
Old 06-28-2009, 06:05 AM
Pipette Filler
Points: 143, Level: 2 Points: 143, Level: 2 Points: 143, Level: 2
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2009
Posts: 14
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: His-tag protein purification under denaturing conditions

Quote:
Originally Posted by zhanghi View Post
Hi,

Would you mind telling me where I can buy this kind of his-tag column?

Thank you very much.
try BioRad or GE
Reply With Quote
Reply

Tags
conditions , denaturing , histag , 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 11:06 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 0.20102 seconds with 16 queries