Debbie Knight

A day in the life: July 17, 2012

In A Day in the Life, research log on July 17, 2012 at 12:36 pm

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab on a particular day. These entries I will call “A Day in the Life…”

So, this week I am learning how to separate proteins by their overall charge. The technique is called isoelectric focusing. It seems like a complicated procedure, so for the moment I’m willing to accept this as a mystical process. As I gain experience working with the technique, I will understand more of the science behind it.  But for now: black box.

I like to learn new techniques. It keeps me on my toes.

So, I’m learning this technique because I have been using Western blot analysis — a procedure which only separates proteins based on their size. I need to find out if what I’ve been detecting is one specific protein or a pile of proteins that just happen to be the same size. One way to determine this is to separate the proteins first by charge, turn the strip 90 degrees, and then separate the proteins by size. So you’re essentially separating the proteins in two dimensions.

Separating proteins by charge. The biggest hurdle for me was figuring out if I was going to do this technique “old school” which apparently nobody does any more (and there’s probably a good reason it has fallen out of fashion). I know one person (my former boss) who has done this technique with thin tubes of glass (called capillary tubes). I have the equipment in the lab to do the technique this way, but everything I’ve read says it is not as reliable as using pre-made strips (new school). Given that I don’t want to spend all my time banging my head on the wall trying to figure out why my experiment isn’t working, I opted for the more reliable “new school” way of doing things.
(Note: this doesn’t mean I won’t be banging my head on the wall, but hopefully it will be for less time)

The next thing I had to do was find a researcher who had the equipment to run these strips and who was willing to let me use it. I used my professional network to find someone.

Then I had to buy a bunch of reagents which I could have spent a couple of days making. Okay, this is where experience comes in. While it might look like I was opting for convenience, I was trying to maximize my chances of success. If I made any one of the reagents I needed incorrectly, it might take me several experiments before I figured it out.

So, I started the experiment yesterday, kits and strips and instruction manual in hand. Seemingly simple: you put your samples in a tray, place the strip over it, and let it incubate overnight. Trouble came in the form of bubbles in my sample — which according to the instruction manual are your enemies. I think I won the battle, but time will tell.

Today I will actually run the samples on the equipment.

And, tomorrow, I’ll find out if it worked.

I’ve got my fingers crossed that it will work! But just in case, I’ve got a couple of consultants on speed dial.


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