Momento Mori...volume II

Throughout this whole process, I've had a very positive beat. And why not? I'm being paid to do science and get a medical education.

Still though, us MSTP kids aren't machines. Amidst all this, I'm still an emotional person. I like people and being around them. Relationships are incredibly important, and I've been so lucky to spend the last year with a very amazing girl.

Yet, in the words of my friend Chester Bennington, "...sometimes goodbye's the only way".

There's a lot on my plate right now with med school proper starting soon and now the big changes in my personal life. Wish me luck. I'll need it.

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.

WAY overdue....sorry folks

Current location: 30 minutes from Spooner, WI, sitting on the patio overlooking the lake, celebrating Amuricuh day (the 4th). Considering the last post was well over a month ago, it may be well to admit that I'm not ready for the responsibility of having a dog, despite wanting one. So this is way overdue, but I've got a lot to talk about (even some science!) - here we go!

The last month has been absolutely INSANE. Fun, busy but absolutely INSANE. The CliffsNotes version, more or less in chronological order:

1. First lab rotation begun and finished
2. Fully moved into the new house
3. New roommate (Erich)
4. Trivia nights on Mondays
5. Eric Clapton live at Summerfest
6. Bought my first car
7. Ramani's engagement!
8. 4th of July reunion with the old roommates (and Kelly and Kim joined us, too!)

So, much has been going on. The big news is the first lab rotation:
I spent about 5 weeks in Dr. Park's lab in the department of biochemistry, studying the MAP Kinase (MAPK) pathway in cancer. Essentially, this is a series of proteins that are responsible for activating the ones below them, creating a chain reaction when the cell is properly stimulated. This translates into instructions for the cell to either replicate, stop dividing (senescence) or die (apoptosis). As one can imagine, this is hugely important in cancer, as the name of the game is get cells that are rapidly multiplying to senesce or, better yet, die. One of the big questions in the field is how this pathway can signal these opposing outcomes with the same machinery and activation. Think about it this way: you have one light switch and by flipping it you can either turn on a fan in one corner or turn on the light overhead. The lab recently found that one of the key proteins in the pathway, ERK, has both catalytic and, interestingly, non-catalytic functions, which may explain why this is possible.

So where do I fit into all of this? Well, I spent the last 5 weeks investigating the function of a drug that is currently in use clinically to induce cell death. They have found the drug to already be very effective in inducing apoptosis in cells that have a mutation in the RAF protein. However, in cancer where this mutation is not present, the drug causes the cells to hyper-proliferate (ruh roh, Shaggy!). So my job was to see if we can use this drug on RAF-normal cells. This is a fancy way of saying I cultured up tons of cells, treated them with the drug, collected protein samples and measured what happened to protein function in the MAPK pathway. Here is an example of some pretty neat biochemistry and ingenuity: how do you separate all of one protein from literally tens of thousands in a total protein sample? Well, it's actually pretty simple, and we do it every day in the lab. First, the cells are broken apart with detergent, allowing us to access the proteins inside. These proteins are then placed into a gel much like Jell-O (but not for eating) and separated by size using electrical charge. So, now the proteins are all spaced out, big huge ones at the top, small ones at the bottom, and everything else between. This sample is then transferred to another apparatus where the proteins are pulled out of the gel and put onto a membrane using, again, electrical charge. Now here is where things get really neat. For anyone who has ever been sick (i.e., all of you), your body developed antibodies for whatever it was that made you sick. Essentially, these antibodies recognize the proteins that are expressed on the outside of the bug you had. Somewhere, someone got a really good idea - these antibodies are VERY good at recognizing a VERY specific protein sequences. So, what if you inject protein, say ERK, into a rabbit? It will develop antibodies that are specific to just ERK! Collect a blood sample from the rabbit, separate the antibodies, and viola, you have an antibody for that protein that you can apply to the membrane! And, it gets even better! So now you have a whole sheet of proteins on the membrane, and all the ERK proteins have antibodies stuck to them. Now get another antibody, say from a goat, that targets rabbit antibodies. While you're at it, add a light-emitting molecule to the antibody and you'll be able to see the proteins on a long exposure camera or undeveloped film. Pretty neat, hey?

So that was 5 weeks, and there are some results that do look a bit promising. It's up to Johnathan, the next rotation student (and fellow MSTPer + past BTRL member) to pick up the project and make it shine. So, good luck to him, hope I didn't leave too big a mess for you, buddy.

So what else? Well, the house is all set up (whew!) and we're getting along great. Back to family dinners with the roommates and FIFA World Cup games on the weekends. Getting back into a rhythm of things, and it's good.

Of course, the really big news is that my sister, Ramani, is engaged! Emmanuel came to Milwaukee earlier this month to spend a weekend and popped the question...she said YES! Needless to say, we're all very excited and wish them the best. Stay tuned for more details and dates.

So that's it for now. And yes, this post was written on the 4th and published on the 17th. I know I'm behind, but whatever. I'll fill you in on Gauld Lab (new rotation) happenings in a short bit. But this should be enough (er, overload) for now.