Hi Nick and everyone, I tried the 0.02% deoxycholate/10-20% TCA method. It works. However, I faced problem in samples with high volume, the resultant precipitate formed a large and unbreakable lump. The lump retained TCA which turned the color of sample buffer to yellow, however, boiling in sample buffer also can't break down the lump. Is there any limitation on using this method, like the amount of total proteins? Would the lump formed by some insoluble components (salts?) rather than proteins? I read some articles mentioned this problem when preparing samples for 2D-PAGE and sonication can improve it. Any other methods can break down the lump since I don't have this apparatus in my lab so that the TCA can be washed away and the proteins are fully resolubilized in sample buffer?
Sincerely, Stan ________________________________ From: Nick Theodorakis <[email protected]> To: [email protected] Sent: Fri, March 12, 2010 12:50:28 AM Subject: Re: Protein precipitation - acetone? On Mar 11, 10:56 am, "Iraz Toprak Aydin" <[email protected]> wrote: > 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? > Acetone shouldn't affect blotting or detection, but depending on the protein, you may need to add several volumes of cold acetone, which may be more volume than will easily fit in one centrifuge tube. Many people have good luck with TCA/deoxycholate precipitation. Add sodium deoxycholate to 0.02% and TCA to 10%. Ice down for about half an hour, then spin hard (e.g., full speed in a cold microfuge for 15 min). Wash the pellet once or twice with cold acetone (or cold ethanol) to remove the TCA, air dry, and resuspend in SDS/gel loading buffer. If the color goes yellow, you didn't wash all the TCA out, but you can add a microliter or two of saturated Tris to bring it back to blue, and be more careful next time. Heat and serve. Nick -- Nick Theodorakis [email protected] contact form: http://theodorakis.net/contact.html _______________________________________________ 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
