Chromotography

A few months ago I  wrote about how to get bacteria to make proteins for you. That was when I was testing out my DNA constructs to make sure that the would make the right proteins for me. I got about a milligram of my protein out of a hundred milliliters of bacteria. I have since used all that up and decided that this time I would make a life time supply of proteins. So for each of my constructs I grew up 2 liters of bacteria. The main difference this made was now instead of being able to isolate my protein with just normal equipment at my bench, this time I got to use the fancy big equipment that makes you feel like a real scientist. 

First the French Press. The french press is not actually the same kind of one that is used to make coffee. This one the main idea is you use a very very powerful hydrolic squisher to force the bacteria through a tiny hole. This breaks them open releasing all their proteins. 

Next FPLC (fast protein liquid chromotography). The proteins I am interested in were cleverly designed back when I was making the DNA to have a molecular form of velcro. This monstrosity of a machine is essentially just a pump that switches between three different sources of fluid. First it sucks up all my bacteria mush and flows it over a cylinder full of the other half of my molecular velcro. My protein with the velcro sticks and the other proteins without velcro don't. After washing off all the extra proteins the machine starts adding increasing amounts of "fuzz" stuff that sticks to the cylinder velcro better than my protein velcro does. this makes my protein fall off and I get it all in a tube. 

Next up Dialysis. Yes this is kind of like Kidney dialysis. I put my protein in a fancy science otter pop bag. The bag is made out of a membrane with little holes in it. The holes are the right size for the "fuzz" to leak out but to keep the protein in the bag. This way I am able to get fuzz free protein. After that I just use a big centrifuge to concentrate my protein and it is ready to use. 

The best part of the process besides getting to use the big machines is that my proteins all glow in the dark so I can tell just by looking at my samples if the machine is working right. If I had to work with normal person proteins I wouldn't know until I got all the way to end if the process had worked.