Hello! I'll be taking an extended break from blogging and temporarily putting a greater focus
Long ago, when I took the applications course for biotechnology, we learned about protein analysis. Similar to DNA analysis, analyzing proteins consists of looking at the order of amino acids. Proteins, however, pose a more significant challenge to analyze compared to DNA. For one, proteins are folded with various charges found along a protein strand, creating folds and helixes. Furthermore, they're vastly smaller than DNA, making it difficult to separate out with the larger pores of an agarose gel. This is where SDS-PAGE comes in, using a similar concept to normal agarose gel electrophoresis.
Let's tackle the first issue: folded proteins with various charges. You may be asking how this is an issue in more detail. If you were to run proteins without fixing this issue, the proteins would run toward both the negative and positive terminals super fast. This is where sodium lauryl sulfate, abbreviated SDS, comes to fix the issue. SDS is a detergent that, when added to proteins, linearizes them and tags the proteins with a negative charge. Linearizing a protein reduces it to the primary structure, and the negative charge ensures that the protein moves only toward the positive terminal. So once you add the detergent to the protein sample and run it through a thermocycler, you can actually run it through a gel.
When dealing with proteins, it's important to consider their size. It's a common misconception that proteins are bigger than DNA. However, it's the opposite. Since proteins are much smaller, the matrix has to be tighter to ensure a good resolution of the bands. While you can certainly increase the agarose percentage to make the matrix smaller, there are limits. A different type of gel made with polyacrylamide is then used. Polyacrylamide, which gives PAGE its name (polyacrylamide gel electrophoresis), is a much thinner gel with smaller pores, allowing for a better separation of smaller particles, which is perfect for proteins.
- Prepare your sample by adding buffer to it
- Heat at 90 C for 5 minutes
- Prepare the gel by removing the comb and tape
- Assemble the cell
- Load the sample into the gel and run
- Remove the gel and stain
It's certainly been a while since I've written an informational post, but that's because I haven't learned anything for a while! My next biology class is DNA/Genetics research, which is next semester. However, I hope you enjoy this throwback. Besides that, I've been hard at work on my essays for college and the manuscript. This has been the most writing I've done in my life, but it's oddly satisfying.