Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum

Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum (http://www.molecularstation.com/forum/)
-   Recombinant Protein Forum (http://www.molecularstation.com/forum/recombinant-protein-forum/)
-   -   Ammonium Sulphate protein preciptation (http://www.molecularstation.com/forum/recombinant-protein-forum/3186-ammonium-sulphate-protein-preciptation.html)

Adelaide_Science 03-27-2008 06:22 AM

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. :thumbdown:

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:cry:)

Cheers
PhD student
Adelaide_Science

admin 04-09-2008 02:27 PM

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:
[Only registered and activated users can see links. Click Here To Register...]


All times are GMT. The time now is 07:48 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 0.09106 seconds with 11 queries