Dear Protein, I Hate You

I would just like to point out how frustrating it is to come into the lab at 4:30am, all set for yet another absurdly long day of CD, have everything else set up, the small molecule all properly diluted... and then, on the very last step of a long, drawn out, overnight stepwise dialysis....

your protein precipitates.

The protein precipitation doesn't happen in the overnight step, so you can show up first thing in the lab at 4:30am, pull out the cuvette to switch it to the new buffer, see the precipitate, acknowledge that the experiment isn't going to happen, say screw it, and go back to bed. It doesn't happen at the next step at 5:30am either, when you will still justify walking back home and crawling into bed. No, no. It waits until 7:00. After you've already washed out the appropriate pathlength cell with SDS and NaOH to make sure it's all super clean and shiny and ready to go. And you've prepared all your standards, and prepped the small molecule in EtOH (no DMSO for CD experiments!) and then diluted it down in buffer to appropriate concentrations, and have everything on ice and all set, and all you have to do is add the protein once it's out of dialysis and you measure the protein concentration. And 7:00 rolls around and you go to pull out the dialysis cuvette and... wait, what's this? What are all these little white specks floating around that weren't there an hour and a half ago? Why, it's my protein, which decided to crash out just now, even though I've done this same dialysis procedure multiple times before without problems.

And thus, tomorrow, I get to try this all over again. Things like this -- the fact that I work with a protein that has to be purified fresh every 4 days, which requires a somewhat complicated dialysis procedure (which apparently no longer works), which cannot be concentrated AT ALL, which hates to be refolded -- this is why it is going to take me approximately 12 years to complete my Ph.D.