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.we...@nottingham.ac.uk> To: meth...@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-boun...@oat.bio.indiana.edu im Auftrag von methods-requ...@oat.bio.indiana.edu Gesendet: Do 11.03.2010 17:03 An: meth...@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.ay...@epfl.ch> Subject: Protein precipitation - acetone? To: <meth...@magpie.bio.indiana.edu> Message-ID: <004401cac133$5b7c0df0$127429...@aydin@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.ay...@epfl.ch> iraz.ay...@epfl.ch ------------------------------ _______________________________________________ Methods mailing list Methods@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@net.bio.net http://www.bio.net/biomail/listinfo/methods Send instant messages to your online friends http://uk.messenger.yahoo.com _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
