Hi Irit and everyone, I heard BSA helps if the sample has very low concentration. I just wonder if the high BSA concentration would affect the later SDS-PAGE. Let say if you need to add BSA to 1mg/mL in 1mL sample. Finally, need to add all the sample for running SDS-PAGE in one well. So there is at least 1 mg of total proteins (Or 1 mg BSA plus negligible amount of proteins from the original unconcentrated sample). There are two problems I may concern. First, whether the lump formed by high concentration of BSA can be dissolved in sample buffer. Second, whether sample with such a high protein concentration would affect the resolution of the SDS-PAGE, esp for protein with small molecular weight. I tried to load 250micro gram total protein extracted from brain lysate and resolve it by SDS-PAGE, however, the resolution is very bad, esp at small molecular weight. My target protein is 4kD and I used 16.5% Tricine gel. The reference for this experiment was mentioned in Lesne S. et al., 2006, Nature, v440, 352-357 and its supplementary methods. But I never get it works.
Best, Stan ________________________________ From: Irit Rappley <[email protected]> To: Nikola Wenta <[email protected]> Cc: "[email protected]" <[email protected]> Sent: Fri, March 12, 2010 2:53:50 AM Subject: Re: Protein precipitation - acetone? Some people add 1 mg/mL BSA to their sample in order to get the total protein concentration high enough for precipitation. Of course, then you're left with lots of BSA in your precipitate too.... On Mar 11, 2010, at 9:27 AM, Nikola Wenta wrote: > 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: [email protected] im Auftrag von > [email protected] > Gesendet: Do 11.03.2010 17:03 > An: [email protected] > Betreff: Methods Digest, Vol 58, Issue 7 > > Message: 8 > Date: Thu, 11 Mar 2010 16:56:05 +0100 > From: "Iraz Toprak Aydin" <[email protected]> > Subject: Protein precipitation - acetone? > To: <[email protected]> > Message-ID: <004401cac133$5b7c0df0$127429...@[email protected]> > 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:[email protected]> [email protected] > > > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > [email protected] > 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 > [email protected] > http://www.bio.net/biomail/listinfo/methods Irit Rappley, PhD The Scripps Research Institute BCC-265 10550 N Torrey Pines Rd. La Jolla, CA 92037 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods Send instant messages to your online friends http://uk.messenger.yahoo.com _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
