Western blot results can either be the highlight of the day, or a scientist's worst nightmare. Unfortunately, it usually turns out to be the latter of the two. After a long hard day of work, nothing can ruin the day more than seeing your western blot results come out blotchy and unreadable. Luckily, there are many ways to prevent and fix a messy blot to ensure you get the best results possible, rather than ending up with something horrific like the image below. To avoid these situations, I have outlined some tips to keep in mind before going through with your western blot test.
Images like these send chills through my spine.
1. Choose the right membrane for your application
PVDF (polyvinylidene difluoride) and nitrocellulose are usually the most commonly used membranes for a western blot test. However, both have different characteristics that you can take advantage of depending on the type of test. PVDF, for example, is considered the more durable choice and can handle re-probing better than nitrocellulose membranes, which are fragile and thus more prone to losing the signal once after being re-probed and stripped. If you find that you’re testing with a large number of proteins, and your test will not require you to strip and re-probe a membrane, maybe try a nitrocellulose membrane. Although they are considered sensitive for having a lower protein binding capacity compared to PVDF, if your testing with a high protein count, making the switch to nitrocellulose should help reduce the possibility of getting a non-specific signal.
2. Follow the washing and detection steps with care.
Washing your tests correctly is an essential step for coming out with the best results. Sometimes, even the smallest changes to this step can mean the difference between high and low-quality blots. For example, Tween-20 is usually the recommended detergent solution to use, but switching to NP-40 (a stronger detergent) instead can be a perfect solution for you to remove high background bands.
3. Always try to use high-quality antibodies
Just like with any “secret recipe” you’ll find at a restaurant, the “secret” usually comes from buying the products with the best possible quality. Unfortunately, there are companies out there distributing antibodies without even having them tested beforehand, causing unnecessary stress for clients. It’s always important to know how to find a good primary antibody supplier.
Sometimes cutting costs is a necessary evil, but it can be a gamble with the results you will get. It’s essential to choose a supplier you can trust, which is why ABclonal goes through strict quality control with all our catalog products. If you’re interested, check out our list of catalog antibodies that can be used in popular research areas ranging from epigenetics, DNA damage/repair, cancer, and immunology.
4. Make sure you’re using the right incubation speed
It’s crucial to always make sure you have the correct shaking speed during the incubation period. The antibody might not be able to bind correctly If you let shake too fast, and making it too slow will prevent the uniform binding of the antibody. Always double-check that your shaking speed is balanced before going ahead with incubation to avoid this silly mistake from taking time away from your research.
5. Recovering a burnt-out signal
If you’ve ever come across a western blot white bands surrounded by black, you know the dreaded feeling of finding out your results ended up with a burnt-out signal. This could mean that you added some extra luminescent substrate around your membrane or transfer cassette that is causing damage to the signal. As a quick fix, try taking out the membrane carefully (maybe with some tweezers) and then just gently shake off the excess liquid. Also, make sure to clean off any substrate left on the transfer cassette with a paper towel without touching the membrane.
A burnt out signal could also be due to high protein concentrations messing up your luminescent substrate. If this happens to your test, the signal can be recovered by titering the protein lysate and diluting the antibodies.
6. Choosing the right blocking buffer.
If you want to ensure you come out with clean blots, choosing the right blocking agent for your western is the way to go. While using milk-based agents is the common way to go, it’s important to remember that these blocking buffers may contain endogenous biotin, glycoproteins, and enzymes that can cause problems with the signal, resulting in high backgrounds. In these situations, you might want to use alternatives such as a BSA blocking buffer. Before doing so, however, always make sure to check if your antibody needs a preferred blocking agent and concentration for it to work well during the test. While a 1-5% blocking solution is often the most recommended, always double-check before going ahead with your test.
We hope that these tips can prove useful for you and your lab partners to avoid another poor and ugly western blot. For further reading and more helpful tips for your research and experiments, check out our curated list of blogs here to learn more about popular techniques and experimental troubleshooting.
Also, If you’re interested in checking out our line of Tanon products for your western tests, click here.