Protein precipitation - acetone?

Stanley Cheung via methods%40net.bio.net (by stanleycheungkk from yahoo.com)
Sun Mar 14 21:39:16 EST 2010


Hi Niko,

Could you describe a bit more about how to make a
acrylamide gel which can compress the protein band (using mini-gel from
Biorad)? I have problem in losing resolution with large sample volume.

Sincerely,
Stan




________________________________
From: Nikola Wenta <Nikola.Wenta from nottingham.ac.uk>
To: methods from oat.bio.indiana.edu
Sent: Fri, March 12, 2010 1:27:40 AM
Subject: RE: Protein precipitation - acetone?

Hi Iraz!
I don't have any clue about acetone precipitation of proteins, but generally, precipitation with saturated Ammonium sulfate solution provides a good means to concentrate proteins and to get them into a save state for short-term storage. Unfortunatelly, you would already need the protein solution to be at > 1 mg/ml in order to get it to precipitate. Thus, in your case this method doesn't seem suitable. You could also try to concentrate the protein with Centricons, but you would rather loose protein to the membrane than concentrate your solution as it is already too diluted. Why not loading maximum volume into biggest possible pockets on a gel with maximum thick spacers? Additionally you could use a acrylamide percentage that "compresses" your protein band, giving you a better signals in WB.
Best, Niko

-----Ursprüngliche Nachricht-----
Von: methods-bounces from oat.bio.indiana.edu im Auftrag von methods-request from oat.bio.indiana.edu
Gesendet: Do 11.03.2010 17:03
An: methods from magpie.bio.indiana.edu
Betreff: Methods Digest, Vol 58, Issue 7

Message: 8
Date: Thu, 11 Mar 2010 16:56:05 +0100
From: "Iraz Toprak Aydin" <iraz.aydin from epfl.ch>
Subject: Protein precipitation - acetone?
To: <methods from magpie.bio.indiana.edu>
Message-ID: <004401cac133$5b7c0df0$127429d0$@aydin from epfl.ch>
Content-Type: text/plain;    charset="us-ascii"

Dear all,



I have to do a western blot, but my protein concentration is very low, and I
have to run a mini gel. So I was thinking of precipitation the proteins. I
have never done this before. Does acetone have a bad effect on the blotting?
Are there any points that I should be careful about?



Thanks in advance...



Iraz Toprak Aydin



EPFL SV ISREC, Station 19

Batiment SV, SV 2540  

CH-1015 Lausanne

Switzerland



Tel: +41 21 693 07 36



e-mail:   <mailto:iraz.aydin from epfl.ch> iraz.aydin from epfl.ch





------------------------------

_______________________________________________
Methods mailing list
Methods from net.bio.net
http://www.bio.net/biomail/listinfo/methods

End of Methods Digest, Vol 58, Issue 7
**************************************


This message has been checked for viruses but the contents of an attachment
may still contain software viruses which could damage your computer system:
you are advised to perform your own checks. Email communications with the
University of Nottingham may be monitored as permitted by UK legislation._______________________________________________
Methods mailing list
Methods from net.bio.net
http://www.bio.net/biomail/listinfo/methods


Send instant messages to your online friends http://uk.messenger.yahoo.com 


More information about the Methods mailing list