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. 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.
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.
It’s crucial to always make sure you have the correct shaking speed during the incubation period. Make sure you’re using the right incubation speed 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.Ĥ. It’s essential to choose a supplier you can trust, which is why ABclonal goes through strict quality control with all our catalog products. Sometimes cutting costs is a necessary evil, but it can be a gamble with the results you will get.
#WESTERN BLOT TROUBLESHOOTING HOW TO#
It’s always important to know how to find a good primary antibody supplier. Unfortunately, there are companies out there distributing antibodies without even having them tested beforehand, causing unnecessary stress for clients.
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. Always try to use high-quality antibodies 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.ģ. Sometimes, even the smallest changes to this step can mean the difference between high and low-quality blots. Washing your tests correctly is an essential step for coming out with the best results. Follow the washing and detection steps with care. 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.Ģ. 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. 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. However, both have different characteristics that you can take advantage of depending on the type of test. PVDF (polyvinylidene difluoride) and nitrocellulose are usually the most commonly used membranes for a western blot test. Choose the right membrane for your application Images like these send chills through my spine.ġ. To avoid these situations, I have outlined some tips to keep in mind before going through with your western blot test. 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. After a long hard day of work, nothing can ruin the day more than seeing your western blot results come out blotchy and unreadable. Unfortunately, it usually turns out to be the latter of the two. Western blot results can either be the highlight of the day, or a scientist's worst nightmare.
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