Monday, July 7, 2014

Western Blots: Lessons from Immunology

Western blots are a very common method of evaluating changes in specific proteins. They rely on some basic concepts from immunology, the study of the immune system. One of the things your body does when you get sick is make antibodies Antibodies are globular proteins that have special arms that recognize one specific item, generally called an antigen. In our immune system, you have antibodies that recognize antigens you've been exposed to. For example, if you've had chicken pox you have antibodies that recognize chicken pox, but if you've never had them, you have no antibodies and are susceptible to catching them. 
GIANTMicrobe of antibody (Y shape) and antigen (red).
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The cool thing is all mammals have immune systems that will react to any foreign particles, which means that scientists can create antibodies to almost any protein they want. Rabbits, mice, rats, goats, sheep and horse are commonly used to generate antibodies for research. To do a successful western, you actually need 2 antibodies. The primary antibody is generated in one organism (for example rabbits) against your target protein/molecule. The secondary antibody is raised against a section of your primary antibody, for example if our primary antibody was made in rabbits, the secondary antibody would be anti-rabbit raised in a different animal, such as goat. Usually, secondary antibodies are also attached to a reporter that will fluoresce when the developing solution is added for easy detection.

The first step in any Western is to obtain the protein from your experimental sample and then separate it out on a gel. Then you place the gel and a special filter membrane into a sandwich of filter paper and sponges. The sandwich is put into an electrical current, the gel towards the negative pole and the membrane towards the positive. This electrical current moves the protein from the gel into the membrane. The membranes act as protein fly paper, trapping them while allowing smaller molecules to pass through small pores.
building a sandwich
We run our Westerns at a very low amplitude in the fridge overnight. This is more for our convenience so we have a place to stop for the day and go home. The next day the membrane is placed into a blocking buffer, for what I am doing I use PBS Milk. PBS is a phosphate buffer to which we add powdered milk. The proteins in the milk will coat the membrane in the areas that your sample protein are not already attached. This step is incredibly critical to obtaining a high quality blot. Without this nonspecific protein coat, the antibodies (which are proteins) would stick to the membrane surface resulting in a lot of background contamination.

Once sufficiently blocked (and this takes hours), the primary antibody is added and allowed to incubate for even more hours. Then a few washes are performed to remove any excess antibody from the membrane. Next comes the secondary antibody step. Then even more washing. Once this is completed, the blot can be developed. We use a chemiluminescent and a special camera to capture the bands. The only areas of the membrane that will glow are those where the primary antibody has attached to the protein and the secondary antibody has attached to the primary antibody.
One of my early PhD Westerns, it's not that pretty.. I will do better!
When we develop them, we tend to look at the blot as a negative, so that the bands are dark and the background light. In the above image, the ladder on the left (dark background) indicates different molecular weights, allowing us to identify the size of our bands. Since we know what protein we were looking for, the ladder confirms that it is the correct size. As you can see in the blot above, the antibody I was using is not incredibly specific, it actually cross-reacts with several proteins from the same closely related family. There is no real difference between the intensity of the bands, meaning that the protein levels were not affected by my experimental conditions.

Now back in my previous grad school life, I was using a more specific antibody that only detected one band. This was pre-chemiluminescent. We had a developing buffer that had to be continually monitored so that the membrane could be quickly moved out of the developing buffer into water or else the background would interfere. As you can see in the blot below, some of the bands disappeared, meaning the protein was decreased in the environmental conditions. 
One of my M.S. blots

The power of Western blots should not be underestimated. It takes a few days, but it is well worth the effort as it provides a clear picture of how the protein levels are, or are not, changing during your experiment.

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