Preparation for an SDS-PAGE Gel

Last Friday (September 6th), I went over to RPI for the first time during this school year! My primary job for the day was preparing for an SDS-PAGE gel. For this gel, we are working with a specific protein of interest.Ultimately, the gel will analyze the bands of the null solution (which does not contain the protein of interest, but contains other host cell proteins (HCPs)) versus the bands of the solution that contains the protein of interest (in addition to the other HCPs). To do this, we are working with different types of protein solutions. First, there are the null solution and the protein of interest solution which are both prepared at 1X and 10X concentration. The different concentrations will ultimately lead to different width bands. Also, there are 10X desalt solutions for both the null and protein of interest. In the original solution, the buffer contains metal ions that can affect how the bands are displayed in the gel, making them appear blurred. In the desalt solutions, the buffer has been exchanged to PBS to eliminate the metal ions and get cleaner bands. Once the gel is run, we expect to see something like the simplified gel below. We expect to get certain bands from the null solution that represent the bands of the HCPs, and we expect to get those same bands from the protein of interest with the addition of an extra band that represents the protein of interest (represented in red in the image).

Today, to prepare for running this gel, I first made the Tris/Glycine/SDS buffer by combining the stock solution with water, mixing the buffer, then putting it on ice to keep it cold (keeping the buffer cold allows for sharper bands in the gel). Next, I made the blue Laemmli sample buffer by combining the stock solution with β-mercaptoethanol (BME). I then labled the solution tubes with the different solutions and added 20 micro-liters of Laemmli buffer to each sample. After each sample was allocated into its respective tube, I denatured the protein solutions in a heat bath for one hour. To prevent the sample tubes from popping open in the heat bath, I wrapped each sample with parafilm (which ultimately caused them to stick together). After the proteins were denatured, I spun the samples, and set them aside to cool. I then set up the gel equipment.

The gel was then ready for JP to run later in the day! I can't wait to see the results when I return to RPI on Friday!