Want to see the end stages of protein isolation?
What does it take to make and isolate protein and what steps are you showing me? (Skip to the pictures if this is not interesting.)
1. Insert the gene for your protein into E.coli.
Comment: The E.coli used are non-infectious, not the disease-causing kind.
Details: The carrier of your protein gene that you are inserting is generally always a "plasmid" or small circular piece of DNA that bacteria sometimes carry in addition to their genome. Plasmids tend to carry extra genes on them that are beneficial for the bacteria. The plasmid will also carry an antibiotic resistance gene on it (not one's that people use - these are for lab use). Once isolates only the bacteria that have the plasmid with the protein gene by spreading them on agar plates with that antibiotic. After overnight growth, only bacteria that have the resistance (and thus the protein gene and plasmid) will survive.
Timeline: 2 days
4. Add a small molecule (IPTG) to the growing bacteria once they've reached a critical mass, triggering them to start making your protein.
Comment: The plasmid gene harboring the protein can be triggered by a small molecule that mimic lactose. If interested, google search the "lac operon" to learn how it works. Relative cell densities are calculated by measuring 1 ml samples of the growing bacteria in a UV-visible instrument that measures how much light is getting through the sample (which grows less when there are more cells), giving one an idea of cell density.
Timeline: 2-4 hours
6. Once the time is up, spin your entire culture of E.coli in a centrifuge to pellet the cells - all of them will be at the bottom in a big mass.
7. Scoop out this mass of cells with a spatula (I know, right? Cookin' up some E.coli, OH yeeeeah) and place them in another chilled tube. The pellet looks like peanut butter but smells nothing like it.
8. Lyse the cells. This means - kill them by splitting them open - and we do this by exposing them to a probe that emits high frequency sound waves that literally vibrate and rip them apart. Pretty violent, eh? Why do this?
Why do this? Lysing the cells is the only way to get the protein that you made out of the cells so you can isolate it.
Comment: That centrifuge is terrifying. It's so high tech, it sings to you when you turn it on. And if you do it wrong, the centrifuge can explode and kill someone. The speed is so fast, that it pellets all the cell debris and heavier cell parts, leaving only the smaller items in the liquid, like ... the protein that you made!
Why do this? To separate the protein you made from all the other cellular debris and junk.
11. Add isolating beads to the tube. These are gel resin beads with a specific molecule on it that will bind your protein. For mine, my protein has an extra "protein tag" attached to it that binds to nickel. My gel resin beads contain nickel and will bind to all my protein, but leave the other proteins in the solution that don't have the tag alone.
12. After an incubation period of 1 hour in the cold room (room that's a walk-in fridge), you can put your bead / protein solution into a column / sieve and let the liquid drip out. Wash your resin beads with the appropriate buffers (to help wash away the proteins you don't want and leave the one bound to the resin behind).
13. Extract your protein. Do this by adding a buffer with a small molecule to the protein that makes the resin beads drop the protein. It will now come out the sieve.
14. Collect it.
15. Put it in a porous dialysis bag and allow any contaminants to diffuse out. It takes a few hrs or overnight.
16. Freeze it for later use!
I am showing you step #16, freezing the protein just after dialysis has been completed!
Note: There are many different ways to make and isolate proteins of interest from cells depending on the type of protein and the cell type one is using to make it. However, this is how I make mine. It's a widely used procedure for isolating several types of proteins.
Labeled tubes - now I'm ready to freeze some protein!
Science Fact: why freeze protein in liquid nitrogen?
Pouring liquid nitrogen!
Commentary: this is quite fun to do
My setup: in the cold room, getting ready to freeze the protein
Commentary: what is this stuff?
At first, the liquid nitrogen evaporates a lot, pouring it into the bin. But once the bin cools down to around it's temperature, it stays liquidy for enough time for me to use it. I just add a little as necessary.