Peter, First of all, thank you for making my own troubleshooting seem like a piece of cake. :-)
I agree with others that an N-terminal tag would be useful, but I would suggest something smaller than GFP. Sounds like you're already working on that. In addition to troubleshooting what it is about your gene that's so hard to express, I like your idea of adding testis extract to the translation reaction. I would use homogenate: just take some tissue (or isolated spermatids if that's possible), mince with a razor if the pieces are big, then homogenize using a Dounce glass and teflong homogenizer. If you really want to make sure that the cells are broken then pass the crude homogenate through a needle. When I used to isolate mitochondria from mammalian cells, that's how I got them out. Pull up the homogenate through an 18 gauge needle, expel through a 25 guage needle, repeat a total of 10 times. 10 min at 10k x g will pellet mitochondria and anything bigger; the sup might contain your missing factor. One more question for you: does your protein go through the secretory pathway? Hope this helps, Irit _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
