My current project is to try to map the NLS and the import factor of my favorite protein. The problem is: everything I have tried has failed and I’m no closer to my goal than I was at the beginning of December. With the holidays, I decided to set up a yeast 2-hybrid library screen and let it cook while I was out of town.
Well….when I got back to work, colonies had grown on the plates and the work was just beginning. I grew the colonies up, assayed them, and then rescued the library DNA. I then grew up these colonies and purified the DNA. After sending the DNA down to be sequenced and re-transforming it to verify the interaction….what do I have? Six proteins that specifically interact with my bait protein but none of which are import factors!
So here’s my question: where the heck do I go next? I’ve done blast searches and pulled up countless papers on these proteins. How do I know what’s relevant and what should get thrown in the garbage. I, of course, don’t want to admit defeat so I’m exploring a few of the possibilities.
The thing that’s really frustrating about all of this is that technically the library screen worked perfectly. I just didn’t get what I wanted.
What I have learned is this: LUCK plays a major role in science and I don’t have any.
