This week, Annie and I had an opportunity to work with one of Professor Didem’s research assistants, Stephanie, to learn Gel Electrophoresis in great detail. This learning process was in preparation for Project 2, where we are trying to identify whether or not there is a difference in protein content between normal eggs and eggs with supplements fed. Gel Electrophoresis, not only with DNA but also with proteins, is a very useful skill that always come in handy for biologists and chemists! So here is my step by step guidance to it:
There were many more steps in preparing for gel electrophoresis than the actual running process. First, we have to create our samples and the loading buffer. For gel electrophoresis, loading buffer usually contains some SDS (a form of detergent that puts on a lot of negative charges onto the sample molecules so that we get the mass-to-charge ratios with the charges of different components being constant and only masses differ), glycerol to weigh down when loaded onto gel, and Tris buffer to maintain the pH. Second, we must our samples by setting the hot plate (with some water so that our plastic micro tubes don’t melt) temperature to 100 celsius and putting our samples in once it reaches the temperature.
While the samples are heating up, we get the pre-made gel (most common ones are agarose gel and polyacrylamide gel; we’re using polyacrylamide gel) that has been refrigerated and insert it onto one side of the box and put in glass on the other side. We must also get a rubber tube on the edges of the box so that the gel gets nicely sealed onto the box.
Then, we pour some running buffer (we’re using 1* Tris-Gly buffer) into the space that holds the gel/glass, as well as into the large space in the box up to the point indicated in the diagram.
Before taking out the comb from the gel, we may use a “cheat sheet” to see the initial positions of the hole more easily. And pour in a little more running buffer so that it fills to the top inside the wells.
After this is all setup, we take the heated sample and pipette out some amount (20 micro liters in ours) and load our samples onto the gel.
We also had a chance to use a sonicator, which is an apparatus for separation of materials using ultrasound. We’re going to use this apparatus to homogenize our egg sample. When we were using this, we had to wear headphones to block some of the piercing ultrasound and we could adjust the frequency and magnitude of the ultrasound shocks. It was pretty cool to see that sound waves could be used in conjunction or replace our use of centrifuge. Annie and I are excited to use all these new skills in the upcoming weeks!