Things are happening


I know it has been quite a while, but I’ve had my fill of grad school-related business and illnesses, but I’m back. Don’t worry, I still have plenty of research to share.

Let’s start out with an oldie, but a goodie. The protyro project has been revived, as we have been writing a publishable-worthy article. The one thing missing from the article was a good gel of -/+ protyro, and this actually proved to be slightly tricky. I produced and purified more protyro protein, utilizing large expressions, but the protein was so pure, the lanes kept filling. This made it seem that there was no difference in protein expression from the minus and the plus lanes. What I did was each time I ran a gel, I would run L|-|+|…|L|-|+, meaning I would run two at a time, but at different concentrations of proteins. I made sure to use a nanodrop to calibrate the – and + proteins, as they expressed at different concentrations. I ended up needing to dilute my protein solutions, as they were so concentrated that they filled up the lanes. After a few gels, I was able to get a pretty looking -/+ protyro for publication.

Reviving my optimism, I restarted my azobenzene project. I felt very confident in the previous synthesis, so I used the last remaining bits to express protein at several different residues of GFP. I ended up doing very large expressions, to maximize my protein. Fortunately I did manage to produce some protein, but it was very very dilute. Interestingly, some of the proteins appeared to be difficult colors. This could be because the azobenzene shifted fluorescence, the diluted protein just looks different from pure proteins, or my late nights were taking a tool. Looking to verify one of these hypotheses, I ran fluorescence scans on the protein solutions, and then ran fluorescence scans on irradiated proteins. For the irradiated steps, I simply just irradiated at 365 nm for 20 mins. To my satisfaction, I found that the fluorescence readings altered, and some changed significantly, especially once irradiated. Though I still needed to prove that this was not just normal GFP, and that my exact azobenzene was put in. I ran a Glaser-Hay test with an alkyne-fluorophore, hoping to test each residue. All of the Glaser-Hays turned up negative. This could be due to two reasons. The first is that my azobenzene was not correctly synthesized. The second is that the protein was too dilute. Either way, I had to redo the synthesis to find out.
A few weeks ago I started the synthesis, but I’ll leave that to another post.

Thanks for reading!