(by irappley from scripps.edu)
Sun Sep 26 02:38:49 EST 2010
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,
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