Nuclear Extraction

This one is almost guaranteed to be confusing. I will do my best. As always, if you have any question, please ask in the comments or an email, and I will try to address it.

I've told you about playing with cells. So what happens after they are transfected? In my case, I transfected them because I wanted them to express a certain protein (my protein of interest is called BORIS). So now, I want access to that protein. BORIS is a protein that binds to DNA. DNA is inside the nucleus of the cell. If you can reach back with me to high school biology, you might remember that a cell has a membrane that is sort of like skin. It lets certain things in (ions, glucose, etc.) and tries to keep certain things out (viruses for instance). It holds the cell together as a singular unit. Inside the cell membrane is the cytosol. This is basically a fancy way of saying the innards of the cell. This includes the all the structures in the cell, the liquid inside the cell, all the proteins that are chilling in there, and a bunch of other stuff that I have no interest in.

I want the nucleus. The nucleus is sort of like a mini cell within the cell. It has a membrane around its outside also, called the nuclear envelope. Inside, is the nucleus, which by our analogy is like the cytosol. It contains DNA and other goodies. So, how do I get the nucleus, and get rid of the cell membrane and the cytosol? The answer is nuclear extraction.

You have probably read that I do a lot of hurry up and wait. That on any given day, I probably don't do more than 2 hours of actual work. That is the antithesis of nuclear extraction. The first time I did a nuclear extraction, it took me about six hours from start to finish. Six hours of actually doing stuff. I have gotten a lot better at it, where my last attempt only took about 4 hours.

First you need to harvest your cells. I have generally done between 15 and 20 plates at a time with my nuclear extractions. I leave most of them in the incubator and harvest only one of my transfected cell types. For example, I am working with mutants of the BORIS protein and I have six that I work with (where one of them is the "wild-type" unmodified protein). I will generally have five plates of any given type of BORIS-protein-expressing-cells. So I take five plates, vacuum off the media (food) from the cells while trying not to actually vacuum up any cells. I then wash the plate gently and vacuum off the wash (again trying to miss the cells). Then, you have to scrape the cells off the plate. This is done with a tool called a cell scraper. It is like a mini squeegee attached to a toothbrush handle. You scrape this along the bottom of the plate, and try to get all the cells to come off and congregate in one place. Then you suck off the cells, and put each plate's worth of cells into a test tube. Repeat this process for all of your plates and you will have harvested about 10^7 cells from each plate.

Next, you stick all those test tubes into a centrifuge, and spin them around at a lazy pace of 300 x the acceleration due to gravity. Hard to believe, but that actually is a lazy pace. The centrifuge can do 14,000 x the acceleration of gravity. The point of this is to pull all the heavy stuff (the cells actually) to the bottom, and leave the liquid separate. Then you try to pipette off the liquid so you are left with just the cells. Then you wash the cells with the same stuff as before, but mixed with some ingredients that will try to prevent the cells from dying/committing suicide (when cells are shocked enough, they will die and send signals to their buddies to kill themselves). Again you stick them in the centrifuge, and again you suck off the fluid, and again wash them. Repeat this previous sentence.

The reason that the centrifuging was at such a slow pace was that we didn't want to rupture the cells. We just wanted to wash them a bit. Now we mix them with hypotonic buffer. The inside of the cell wants to be at equilibrium with the outside of the cell. This is generally done by controlling the amounts of ions that pass through the membrane. This will then change the amount of water in the cell by osmosis. The hypotonic buffer will make the cells swell up.

You let the cells swell for a little while then add detergent. The detergent will break apart the cell membrane. Stick these broken up cells in the centrifuge, and the liquid portion will be the cytosol. Pipette that off and if you are interested in the cytosol you can store it for use later.

I, however, am interested only in the nuclear fraction. I just throw away the cytosol at this point. Now, you add nuclear extraction buffer (creative name, huh?). You shake/mix vigorously for a few seconds. Let it sit for a few minutes. Shake/mix vigorously for a little longer. Let it sit for a few. This should have turned the nuclear envelope inside out (I'm not so clear on how it does this...). Then you centrifuge at 14,000 x gravity. The liquid portion of this is the nuclear fraction, and the pellet (the non-disolved stuff at the bottom) is cell membranes and nuclear envelope and other stuff that we don't want. We pop the nuclear fraction into fresh tubes, and stick it in the -80 C (otherwise known as really frickin' cold) freezer, and we are done.