Update Time!

Time for an update! This is for two really important reasons: 1) it's a desperate attempt to give this blog some much-needed attention and 2) I'm done with my molecular work for the day and I'm riding a wave of productivity. That and I can't really leave for another hour, so hey, why not.

So the last post was mostly about the work in the old lab, so time to talk about the new one.

As I mentioned before it's an auto-immunity lab, so let's talk a little bit about that. Obviously, people have an immune system so that we can fight disease and foreign bodies that have some how entered ours. Part of this exceptionally intricate system are the B-cells, who recognize an antigen (a molecule that the cell uses to recognize the things around it). If it recognizes the antigen, it engulfs the whole body and destroys it. In a normal person, 75% of the B-cells that are made recognize (and hence, target) the body itself! So the big question is this: what happens to those 75% cells? Turns out, they undergo B-cell anergy, where these harmful cells are essentially inactivated. In a patient with an autoimmune disease, this doesn't occur, or does not occur to the same extent that it does in other people. Solve B-cell anergy, solve autoimmune diseases, or so is the hope.

So where do I fit in with all this? Well, as I said I'm immunostupid, but I AM useful in that I know biochem. So, I'm working on a project to help the lab out by creating and optimizing a high-throughput western blot assay - like what I did last month but in a plate instead of an actual gel. Here's how it goes down:

1. Add cells to a standard 96-well plate and let them stick to the plastic
2. Fix the cells so they're not moving around, replicating and will stay in one place
3. Punch holes in the cell membrane (permeabilize them) so reagents can enter
4. Add reagent to reduce non-specific binding (antibodies aren't perfect, so they sometimes bind to stuff they shouldn't....bad antibodies!)
5. Add the primary antibody (the one that recognizes your protein of interest)
6. Wash it out and add the secondary antibody (the one that recognizes the one that recognizes your protein and has a fluorescent tag on it)
7. Scan it in an infrared imager where the secondary will light up

It should look something like....this! (Each red circle is one well and the red is the protein lighting up)

So, let's compare regular westerns vs. in-cell westerns, shall we?

	In-Cell	Regular
Time	Big props to ICW on this one. Since you don't have to lyse the cells, run the gel, transfer to a membrane and THEN start working, it's a huge timesaver	The pokey puppy of the two...
Scale	Again, points to ICW. You can run 96 wells at a time in one go, and that's a lot.	Regular western, not so bad. But, you can only run ~10 samples per gel, 2 gels per run...that's a TON of gels before you start matching ICW.
Cell Number	ICW is designed for low cell counts. The image about was done with 250,000 and 1,000,000 cells per well.	Regular western requires a ton of cells in order to produce enough cells for a sizeable cell lysate and high enough protein concentration. Think 5,000,000+
Antibody	Sorry ICW, you lose this round. Antibodies are really expensive, so the less you have to use, the better. Normal concentration here is between 100-200x dilution.	Depending on your protocol and the protein, could go as low as 5000x dilution, although I've personally only gone as low as 2500. Still, that's about 200x less antibody used each time, and that saves ca$h.
Specificity	Before you can really run an ICW, you have to run the corresponding western normally (gel/membrane western).	In a regular western, you separate out the proteins by size. Since you do that, you can see if you are getting non-specific binding really easily (i.e., the gel lights up way away from the protein it's supposed to bind to). You can't do that in ICW, so you have to do one of these first to know that your antibody is legit.