The Biochemist’s Lab Notebook
At first glance, it may seem clear that scientists do science, much like an engineer engineers things, a referee referees things, and a janitor—well, forget that last one. Unfortunately, there seems to be a rather severe disconnect between what scientists tell their family versus what they actually do. The goal of this feature is to try to break that barrier down.
A few times each month, I will post an explanatory version of what I’ve done in lab that day. Most of these will be biological procedures because I am a biologist, and most of them will be explained in layman’s terms, hopefully with hyperlinks to more explanatory information if desired. The goal is to give nonbiologists a window into what “I work on cancer” truly entails and how a person can spend their entire career on those four words.
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A beginner’s guide to neurotoxic jelly and high voltage
Well, I suppose it’s time to kick this feature off with a… well, hopefully not with a bang because “bang” isn’t a good sound to hear in a laboratory. Today I began what’s known as a Western blot. It’s a procedure designed to separate proteins by size, which presumably will allow you to identify them. Usually, some manipulation is done to the proteins beforehand, but we’ll save that for another post.
The technique basically exploits high school geometry. First, the proteins move through the gel in one direction, let’s call it “vertical.” This separates them by size. Unfortunately, gels aren’t permanent, so we need a way to store this vertical sorting. We do that by shifting the proteins horizontally onto paper (but it’s very fancy). Their vertical position stays the same so we can still see their size, but they move from a fragile gel to a (relatively) rugged paper.
So, first I have to make a gelatin that will separate these proteins. The “gel” is only a few millimeters thick and is mostly made of neurotoxin, bubble bath, and water. It remains liquid almost indefinitely until I add a catalyst, as which point it becomes almost the consistency of Jello Jigglers. Delicious, especially since the neurotoxin’s neutralized in the process.
Then we put our protein samples on top of the gel and run an electrical current through the whole mess. The nature of the gel means all our proteins get pulled toward a positive charge, but this gelatin is kind of like a maze of sewer tunnels. Slim Eddie is going to have a lot easier time running through the maze than Jimmy the Behemoth (apologies to anyone who actually bears those names). 3-4 hours later, Eddie’s near the end while Jimmy is still mired in the middle, searching for tunnels he can fit through. In other words, our intrepid aqueductal adventurers have been sorted based upon size, just like our proteins. Then comes more electricity!
To “transfer” the proteins from the gel to the “fancy” paper, we lay the gel on top of this explosive paper (told you in was fancy!) and apply another electrical current in a different direction (horizontally). Besides launching projectiles, it turns out that this paper is really good at sticking to proteins too. So all of the protein stays where it was in the gel vertically, but after an hour, it moves horizontally from gel to paper.
Finally for the day, I “blocked” my paper (commonly called a “blot,” hence Western blot). This is done by adding milk or something else filled with protein to the blot and it basically coats all of the unoccupied areas. I like to think of it as sprinkling glitter on my 1st grade art project that was drowning in glue. Remember kids, sticky things are bad in your backpack. So, to carry the analogy further, my “blot” is a piece of construction paper that’s covered in glue. My “proteins of interest” are macaroni, and the milk is a fine layer of glitter. So really, today I got to be a very sophisticated 8-year-old for a grand total of about 6 hours.
Next time, I’ll explain how we tell the difference between the elbow macaroni and the wagon wheels because this is critical when you study cancer.
–Zach Bohannan
Tags: blot, gel, lab notebook, proteins