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
