Nobody wants to end the day with a failed experiment, wasting precious time and money, but let’s face it, it happens. Western blotting is just one of those experiments that requires good technique as well as high quality reagents. It may seem like a good idea to cut costs by purchasing economical materials, but this could end up costing you a small fortune if your Western blot fails. When it comes down to it, the best technique won’t make up for inferior equipment, reagents and materials.
For the labs running Westerns routinely, things typically run smoothly. Unfortunately, it tends to be the occasional blotters who run into the most trouble. This can be very frustrating since there are many points at which something may have gone wrong, making troubleshooting for the unexperienced researcher quite a frustrating experience. Those who have mastered the art of Western blotting know that high quality reagents and materials are critical for success. Those materials are worth investing in because the cost of a Western blot failure may be higher than you initially thought.
Loss of Precious Sample
The work leading up to a Western blot can often take months and may have only yielded miniscule amounts of your precious sample. A poor quality Western will destroy this highly coveted sample mercilessly. The true cost of your failed Western should include the cost of producing that Western blot sample. This cost may actually be quite high if you take into consideration the time and money that was spent purchasing tissue samples, primary cell lines, cell culture materials or room and board for small animals. This can easily cost thousands of dollars, so it is well worth the extra effort to ensure that everything is working as expected before diving in.
A Western blot is a bit of a nightmare to troubleshoot. Was it the sample? The transfer? The blocking buffer? The antibodies? I could go on, but you get the idea. Setting up control situations to verify an issue with any of these parameters is often quite challenging. Trying to find the culprit may take several rounds of testing, which will end up being very expensive due to the high cost of primary antibodies. This process will also be very time consuming, not to mention extremely frustrating.
So, the moral here is to put some extra thought and care into designing your next Western blot experiment to save yourself time, money and quite possibly your sanity. Start off by carefully scrutinizing antibody data provided by the supplier. Next, verify that the supplier’s data is representative of the antibody’s quality by searching antibody databases. Then, ensure that all buffers are made with high purity water and chemicals, as well as filtered to remove particulates. Use low auto-fluorescence membranes for the lowest background. Finally, make sure that all your equipment is clean and in good condition. Once you have your reagents, materials and equipment under control, it is wise to verify your method with positive and negative control samples before experimenting with precious samples.
Blotting Sponge Pads
Flashblot Transfer Buffer
AdvanBlock-Chemi Blocking Solution
AdvanWash Washing Solutions
WesternBright ECL HRP Substrate