WIestern blotting is a technique used to confirm the presence of a particular protein in a sample containing other proteins. Each western blot experiment uses a specific antibody against a specific protein. These antibodies bind to the protein of interest and help quantify it later.
Western blotting is a common research technique in molecular biology, immunology, and related disciplines. For students looking to pursue careers in these fields, Western blotting is a critical concept to master. But students learning about it for the first time might find the lessons intimidating. Thankfully, there are many ways to make it more engaging and approachable. Read on to find out how.
Image from Labster's Perform a Dry Western Blot virtual lab.
First, let’s look at why students find it challenging to learn Western blotting. Here are the top three challenges according to both students and teachers.
Before Western blotting can detect proteins, they must first be extracted from cells and denatured. Protein extraction can be complicated, depending on the kind of cells the proteins come from. Once the proteins are collected, they need to be denatured to remove the various 3D shapes they are normally in. Otherwise, the Western blot may not provide proper results. Denaturation may involve toxic reagents like β-mercaptoethanol, which can be fatal if inhaled. Lab safety is key when working with Western blots.
Sample preparation is already tedious, but the Western blotting procedure takes even more time and effort. Many times, Western blotting experiments can take an entire day or more to complete. If a slight mistake is made in the middle of the process, students will have to repeat the entire procedure.
Usually, antibodies that target specific proteins are quite accurate for Western blotting. However, it isn’t always the case that students have positive results for their Western blotting experiments. Several things can go wrong, such as incompletely denatured proteins or insufficient amounts of the protein of interest. If students find that their Western blot is negative despite the protein of interest in the sample, they will have to troubleshoot their procedures. Troubleshooting may be even more time-consuming than the Western blot itself.
Based on students' difficulties when studying Western blotting, here are five tips for educators to make the topic more interesting. Each piece of advice addresses a particular challenge that students face.
Western blotting was developed in 1980 by W. Neal Burnette. While working with retroviruses at the Fred Hutchinson Cancer Research Center in Seattle, he developed the technique. He submitted a paper to the journal Analytical Biochemistry, which the journal rejected at first. The paper was all the rage among molecular biologists at the time, though; because of its popularity, the journal eventually published Burnette’s paper.
Fun fact: Brunette also served in the US Army in the 1980s as a field medic and an expert in infectious diseases. He retired from the Army in 2005.
Another fun fact: Brunette wanted to pursue a career in acting when he was younger. But his wife convinced him to pursue science, so he did. Thanks to that, we now have a powerful technique not only useful in research but also in medicine for diagnosing conditions like HIV-AIDS.
As mentioned earlier, Western blotting can be used to diagnose HIV-AIDS. In particular, the technique is used to detect antibodies against HIV. If there is a lot of virus in a patient’s body, there would be many antibodies, so a positive Western blot would indicate the patient is infected with HIV.
If students can appreciate this key application of Western blotting, they can understand this topic even further.
Students must first have a proper understanding of key fundamental concepts before studying Western blotting. Here are some of the basics that students need to understand this topic.
Proteins
Proteins, the building blocks of life, are synthesized in all living cells. Humans have tens of thousands of types of proteins, which are all constructed from 20 amino acids. Multiple amino acids connected by peptide bonds form a polypeptide.
However, a polypeptide is not the same as a protein. A functional protein is not just one polypeptide chain; it is one or more polypeptides precisely folded into a unique molecular shape. This specific protein structure determines its function.
Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gel electrophoresis (PAGE) is a technique used to separate proteins by size. Since proteins have a 3D structure, it must first be denatured to make them linear. In turn, they will move through the gel more easily according to their length. This can be done by adding sodium dodecyl sulfate (SDS), which is a detergent that unravels proteins to make them linear. SDS is also required to charge the proteins negatively. That is why this technique is commonly known as SDS-PAGE.
When the proteins are linearized and have a negative charge, they can then be loaded onto a polyacrylamide gel. When an electric field is applied, the negatively charged proteins will travel through the porous polyacrylamide toward the positive pole located at the end of the gel. Longer proteins will travel slower than shorter proteins. This is how proteins can be separated by their size.
Electrotransfer
Polyacrylamide gel is an excellent material for separating proteins but is unsuitable for antibody detection. Therefore, proteins in polyacrylamide must be transferred to a solid support membrane—usually made of nitrocellulose or polyvinylidene difluoride (PVDF). The transfer of proteins to a membrane utilizes an electric field perpendicular to the gel's surface.
Protein detection with antibodies
Detection of the protein of interest is made possible by using specific antibodies. Before antibodies are applied to the membrane, a blocking solution, typically 5% bovine serum albumin (BSA) or de-fatted dried milk, is added to prevent nonspecific protein binding.
Generally, two antibodies are used in Western blots: primary antibodies and secondary antibodies. The primary antibodies have a unique binding site that can recognize specific proteins. After the membrane is incubated with primary antibodies and unbound primary antibodies are washed away, the secondary antibodies are added. The secondary antibodies will bind selectively to the primary antibodies. The secondary antibodies serve not only as a carrier of the detection label but also as an amplifier of the emitted signal. The label attached to the secondary antibodies is usually a biotin or a reporter enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP).
Protein visualization
Chemiluminescence is the most widely used detection system in Western blotting. In chemiluminescence detection, an enzyme-linked secondary antibody is used to convert a substrate to one that produces light emission. The light emission is detected either on a camera or photographic film.
The Western blot result is considered semi-quantitative—meaning it provides a relative comparison of protein levels, not an absolute measure of quantity. Thicker bands denote higher quantities of protein.
Students who have no experience in molecular biology may find this topic daunting. For this reason, it’s important to use actual experience to build their confidence. It’s helpful to have students practice the techniques used in Western blotting, like SDS-PAGE and sample preparation.
Once students get the hang of the basic techniques, they will be equipped to handle actual experiments in the lab. They will already be familiar with routine methods and be more confident in performing Western blotting experiments.
Video demos and interactive simulations prove useful if lab work cannot be done. Simulations like those found in Labster can help students have the necessary skills they need before embarking on actual lab work.
Virtual lab simulations are excellent tools for teaching Western blotting. Labster is determined to deliver fully interactive advanced laboratory simulations that make use of gamification elements like storytelling and scoring systems while exposing students to an immersive, realistic, 3D environment.
Check out the simulations Western Blot Transfer: Prepare for protein detection and Perform a Dry Western Blot at Labster. This virtual lab allows students to perform Western blotting in a safe, computer-generated environment. With this, students will gain the confidence to eventually perform the procedure on their own in an actual lab.
The image below is an example of what students can explore in the simulation.
Please take a moment to check out Labster’s Virtual Labs called Western Blot Transfer: Prepare for protein detection and Perform a Dry Western Blot, or get in touch to find out how you can start using virtual labs with your students.
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