Western Blots

I’m learning how to western blots this week. Western blots are what we use to detect protein levels in cells and is a very common procedure in our lab. It’s not that hard but it takes 2-3 days to do one blot and there are a lot of steps to do. First, we have to collect the cells and lyse them. Most proteins are in the cytoplasm of the cell so we just have to break the cell membrane and collect what’s inside. If we do this with hundreds of thousands of cells we can collect a solution containing a detectable level of protein that we can then analyze. We then take the protein from the cells and load them in a protein gel. The gel is made from an acrylamide mix and can contain several different things. The one we use most is called a SDS PAGE gel. SDS is a type of detergent that changes the protein structure so that the three dimensional structure of each protein has no effect on how it runs in the gel. SDS also puts a negative charge on the proteins. This is key because it allows us apply a current through the gel that allows the proteins to travel down the gel toward the positive cathode. This separates the proteins by size; the larger proteins travel less distance while smaller proteins travel farther. After the gel is run, you have to transfer the proteins from the gel to a special membrane. I’m not sure what’s so special about the membrane (it just looks like a thin piece of paper) but that’s what we use. After we transfer the proteins to the membrane, we apply the primary antibody, which is protein specific so that it will only bind to one protein (or a couple of proteins if you’re unlucky). We then apply a secondary antibody which binds to the primary antibody. Attached to the secondary antibody is an enzyme called Horseradish peroxidase (it’s found in the roots of horseradishes hence the name). Horseradish peroxidase oxidizes a compound called luminol and releases light when the reaction occurs. We capture this light in a dark room with very little other light using a piece of film similar to the type that x ray images are printed on. We then have a machine that develops the films for us, similar to how people did before digital photography. We should see a small black band indicating that the protein is there if the process works.

Western blots aren’t used that often as a data collecting tool because they aren’t qualitative. You have no way of knowing how much protein you actually have; all you can do is compare your samples to other samples. You don’t get any numbers to compare. It can be very subjective. Western blots are mainly used to confirm that you have or don’t have protein in your sample. When we do a gene depletion like I mentioned in a previous post, we can check for the protein of interest by doing a western blot.  If we don’t see a very dark band compared to a control sample without the gene depletion, we can prove that our gene depletion was successful.

Our lab does westerns a lot. They aren’t too hard or too complicated but they are very important. They can be tricky sometimes depending on the protein but I haven’t run into any protein that is hard to see yet. I will at some point but hopefully not too soon.