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

Recombinant Protein Forum Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


Ammonium Sulphate protein preciptation

Ammonium Sulphate protein preciptation - Recombinant Protein Forum

Ammonium Sulphate protein preciptation - Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 03-27-2008, 06:22 AM
Pipette Filler
Points: 1,007, Level: 18 Points: 1,007, Level: 18 Points: 1,007, Level: 18
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2008
Posts: 10
Thanks: 0
Thanked 1 Time in 1 Post
Unhappy Ammonium Sulphate protein preciptation



I am currently tring to isolate three His-tagged proteins (all separately) using ammonium sulphate precipitation followed by loading and elution from a His-Trap Nickel Column (GE). The three proteins are 37.67, 26.34 and 24.12 kDa (+ Hist Tag).

I am following a protocol to isolate the 3 proteins which had previously been used for the 26.34 kD protein in another species, using 40% then 60% ammonium sulphate saturation. However this does not work for the other two proteins , and i can't get them pure enough to do follow on experiments (eg DNA Binding Assays) without the confidence that the other proteins are actually the ones binding the DNA.

I was wondering if ther was a quick way of working out the saturation needed based on the protein properties (eg size, pI, acidic or basic), instead of just doing increasing amounts of saturations and running these on gels to see if my protein is still there (therefore lots of extra work)

Cheers
PhD student
Adelaide_Science
Reply With Quote
  #2  
Old 04-09-2008, 02:27 PM
admin's Avatar
Administrator
 
Join Date: Nov 2005
Posts: 1,418
Thanks: 883
Thanked 68 Times in 58 Posts
Default Re: Ammonium Sulphate protein preciptation

Hello,
thats a good question but requires a tough answer.

Have you tried the equation:

g = 533 (S2- S1)
------------------
100- 0.3 S2

where S1 is the starting concentration and S2 is the final. g is AS in grams


Thats an estimate however you can also try optimization using aliquots of your protein (however you need quite a lot 20-50mL usually).

This book was helpful:
http://books.google.com.au/books?id=...l=en#PPA138,M1
Reply With Quote
Reply

Tags
ammonium , preciptation , protein , sulphate


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 - 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
New Saccharomyces Sequences 09/10/03 SGD Sequences Yeast Forum 0 09-11-2003 01:33 AM


All times are GMT. The time now is 11:33 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 0.16013 seconds with 16 queries