Method of the Month — Western Blotting
September 2021
For our first “gel-based” method, we will be covering Western blotting, a technique that allows the identification and quantification of specific proteins in a sample. If you missed our introductory section on gel electrophoresis, click here to read that feature first.
So let’s say you have a sample full of proteins that you’ve extracted from some cells. That’s great, but you’re not interested in all of the proteins (and there are quite a few). You just want to know about one single protein. Protein names are often long and complex, so for simplicity’s sake, let’s say our protein’s name is Dave.
A relatively simple way to find out whether or not Dave is in your sample is to run a Western blot, one of the most commonly used methods for protein analysis in biology.
Prepping your sample
The first step in this process is a standard gel electrophoresis experiment, but before you do this, you have to prepare your sample for analysis. There are several different ways to prepare your protein sample, but we can’t cover all of them here. We will look at one of the most common methods of preparation: treatment with Sodium Dodecyl Sulfate (SDS).
While the chemical name looks intimidating, you may be surprised to learn that SDS is really just soap…that’s all. If you check the back of most soap bottles you’ll likely see this ingredient or a similar chemical. But, now you’re probably wondering, “Why soap?” A drawing will help explain.
This is a simple schematic of an SDS molecule (click here for the chemical structure). The blue head is negatively charged, and the tail is hydrophobic (meaning it has no charge). When a group of these little SDS molecules are put in solution with our friend Dave…
The SDS molecules attack our protein (along with all other proteins in the solution) like a group of hungry piranhas, and unfold it.
Remember, a protein is just a chain of amino acids that’s folded into a 3D structure with positively charged, negatively charged, and hydrophobic regions. The SDS piranhas disrupt the 3D structure of proteins by binding all over the protein chain, the negative heads interacting with positive parts of the protein, and the hydrophobic tails interacting with similarly hydrophobic regions in the protein.
Why would you want to do this? Well, if you look at denatured Dave, he’s covered in negative charges. Treatment with SDS removes many of the variables in protein structure (shape, charge distribution, etc.) by turning your sample into (essentially) a bunch of negatively charged strands of different lengths. Since all the proteins in your solution are coated in SDS, they’re just long anions, which means that they’ll run readily towards the positive electrode in our gel electrophoresis experiment, and the speed at which they move will be dependent on their size only. Brilliant, right?
Next, you take your protein sample and run it in a gel electrophoresis experiment, separating all the proteins by size. Now, you have another problem. Your sample may contain hundreds or even thousands of proteins, and separating them out by size alone is not enough to find your particular protein. The next step of the Western blotting method solves this problem.
The Blot
As great as gel electrophoresis is, it has a few downsides. The gels used are flimsy and the separated proteins inside them are not easily accessible for further analysis. To get around this issue, the samples are transferred from the electrophoresis gel to a blotting membrane. This membrane is sturdier, thinner, and more permeable than the original gel. To complete this transfer, the gel is sandwiched into an apparatus that runs an electrical current through it, moving the sample from the gel to the membrane. If this sounds like gel electrophoresis, just in a different direction, that’s because it basically is. You’re moving all your protein samples from the gel, to the blotting membrane using electricity.
Now that everything is in the membrane, it’s time to find our protein. This is accomplished using antibodies, which are excellent for binding to things specifically. You can think of this antibody as “anti-Dave,” meaning it will bind to Dave and no other protein in the solution. First, you wash your membrane with (funny enough) milk protein to keep your antibodies from binding non-specifically to anything in the membrane. Next, you place your membrane in a solution containing the antibody that will bind to some sequence in our protein Dave. This antibody is known as the primary antibody. You then wash the membrane to remove any leftover antibody that isn’t bound.
Great! We found Dave. But, we also need a way to indicate that our primary antibody has found Dave which is visible on our gel. For this, we have to use a “secondary antibody” that will both bind our primary antibody and produce a signal for us to detect. Just like with the primary antibody, you wash the membrane after placing it in the secondary antibody solution to remove any unbound antibodies.
There are many ways to make a secondary antibody “produce a signal”. One of the most common ways is to attach an enzyme to the antibody that allows it to convert some chemical into a colored product. That means that once you pour that chemical onto the membrane, anywhere the secondary antibody is bound will by stained a certain color. To see some pictures of membranes from completed Westerns, check out these photos.
And there you have it, the basics of a Western blot! By first separating proteins according to size, then transferring to a membrane, and finally tagging with antibodies, you are able to detect the presence (or absence) of a particular protein in a complicated mixture. There are many modifications and variations on this basic framework, and I’d encourage you to think of some ways to tweak this method so it could be used for new applications. We hope you enjoyed this feature, and we’ll see you next month for our next gel-based method!
