What are proteins and how can you look at them?

We’ve covered a number of topics in the Science Simplified blog and it’s time to learn about another important topic: proteins! Proteins are important for the body to work properly: different proteins can perform a variety of different functions to help our body function. For example, different proteins have different roles, such as to:

• Provide structure to a cell: like the bricks of a building
• Act as a messenger: convey important messages between cells, tissues, and organs
• Cause biochemical reactions throughout the body: an example is the enzyme lactase that help the body digest lactose
• Transport and store nutrients: proteins can carry substances around the body through the bloodstream while others can act like a gate to allow other proteins through
• Improve immune health: proteins can help fight infections
• Maintain fluid balance: proteins make sure your body has enough fluid in the proper places

These are just a few of the examples of different roles proteins play in the human body. Many diseases are caused when proteins don’t work properly. For example, in SLC13A5 Epilepsy (Citrate Transporter Disorder), changes to the SLC13A5 gene makes it so the NaCT protein does not work properly. (For a review on genetic mutations, check out our previous Science Simplified blog post). When working properly, the NaCT protein transports citrate from the extracellular fluid into the cell. This is important for a cell to have appropriate energy levels. Scientists are still working to understand why this change causes such a severe disease.

There are a variety of reasons that could lead to a protein not working properly. Sometimes the protein is in the wrong spot, sometimes the protein is not in the right shape, or sometimes the protein is not around at the appropriate time for it to work right.

To learn more about a specific protein, researchers may ask the following questions:

Then, when a specific protein is associated with a specific disease, researchers can study a disease model and ask:

• Where is the protein located?
• How much of the protein is being made?
• Is the protein expressed at the right time?

How can scientists visualize proteins?

One of the ways scientists answer these questions is by visualizing proteins. This can be accomplished by using immunohistochemistry, or IHC for short. Another name for IHC is antibody staining, because antibodies are used to label proteins in a cell or tissue. This can lead to some very important information! For example, IHC can determine which types of cells express certain proteins. Let’s use Protein X as an example. Is Protein X found in neurons? Is Protein X found in a specific type of neuron? And by looking for protein expression at different stages of development, this can help determine when Protein X is expressed. Is Protein X found only during early development, adulthood, or is the protein expressed throughout life?

How does IHC work?

Let’s walk through a general overview of IHC. One of the first steps in IHC is to prepare a tissue of interest. Let’s say we are studying a mouse. If we want to check if Protein X is found in the brain, we would remove the brain, use chemicals to preserve the brain, and then use a machine to slide the organ into thin slices. This could be similar to slicing a piece of meat into deli meat slices. Each tissue slice can then be used for IHC!

After the tissue is prepared, the tissue goes through a series of washes with specialized reagents to recognize a protein of interest, in this case Protein X. These washes often take 2 days! Scientists may use fluorescent IHC where the end result shows cells that fluoresce a specific color, such as green.

Depending on the IHC targets, sometimes scientists can label different proteins each with a different color. This allows scientists to look at multiple proteins at once by using multiple colors such as green and purple. For example, in the image below, you can see multiple colors. Protein X is labeled with green and Protein Y is labeled with purple.

When a cell expresses Protein X, scientists may describe this as “Cells are positive for Protein X.” If a cell does not express a specific protein, a scientist may describe this as “Cells are negative for Protein X.” Sometimes a cell expresses both proteins (both Protein X and Protein Y). A scientist may describe this as a “double positive cell.” Scientists may then quantify how many cells are positive for Protein X, how many are positive for Protein Y, and how many are double positive.

If scientists zoom in even further, it may also be possible to see which regions of a cell the protein is found. For example, some proteins are only found on the edges of a cell called the cell membrane, or maybe they are only found in the nucleus of a cell. The localization of a protein within a cell can also provide insight into its function.

Visualizing protein expression using IHC can be extremely useful and takes a lot of work. Just like taking a photo with a camera or with a camera phone can take time to get the perfect shot with everything in focus, the same goes for using a microscope to take pictures of tissue and cells. Once all the images are acquired, then the quantification can begin.

Conclusion

Hopefully this article explained a little bit more about how scientists visualize proteins using IHC. Our goal is that the next time you are reading an article and read about staining or see an image with IHC you might be able to understand it a little bit better. Send TESS Research Foundation an email if you have other questions!

This article was written by Tanya Brown, PhD. Tanya is the Scientific Director at TESS Research Foundation. Images were produced using BioRender.com.

Is there a topic you want to see covered in Science Simplified? Let us know by emailing tanya@tessfoundation.org.