Since its inception in 1979, the Western blotting process has remained relatively unchanged. It was and continues to be a deceptively technically challenging method. When it works well, it is a fabulous tool. When it doesn’t work, the researcher is often left scratching their head. Is it my sample? My gel? My transfer? My antibody? My buffers? With so many places to go wrong, it is often challenging to tease out which step is causing the problem. In this post I’ll talk about three of the most common ways to fail and strategies to recover.
I. Nonspecific banding
When I see nonspecific banding on my blot I usually place the blame on the primary antibody. Unfortunately, all commercial antibodies are not created equally, even though they all cost an arm and a leg. When nonspecific bands show up on your blot, the first step is to decrease the amount of primary antibody that you are using. If this doesn’t solve the problem, decrease the amount of lysate that is loaded. If both of these quick fixes don’t resolve the issue it’s time to order a different primary antibody. Before rolling the dice again, choose an antibody that has been validated and published by peers. Search engines such as CiteAb are a great place to begin your search. https://www.citeab.com/
After you have found a new antibody to test, don’t forget that many suppliers now offer trial sizes. Before spending $300-500 on an antibody, make sure it works. Placing another order may be a major inconvenience but the savings will be well worth it!
II. High background
There’s nothing worse than a Western blot that looks more like a Rorschach Test! When this happens it is best to take a step back and prepare new buffers for every step of your workflow as well as swap out any supplies that are re-used, such as sponge pads. For Western blots with the cleanest background, don’t forget to filter buffers before use to remove particulates. Additionally, using low fluorescence membranes will also help to produce the lowest background for both chemiluminescent and fluorescent applications.
III. Weak or no signal
This situation is a little more complicated as the possibility of failure may be coming from a few different sources. Assuming your gel electrophoresis and electrotransfer processes are acceptable, as confirmed by total protein membrane staining, take a look at these likely culprits. (1) The primary antibody may lack sensitivity, making it difficult to detect your protein. (2) The abundance level of target in your lysate sample may be too low to detect. (3) Your blocking buffer may be masking the epitope on your target, decreasing the sensitivity of your Western blot.
For Western blots with signal that is weak or non-existent, Advansta has developed AdvanBlock-Chemi. This novel blocking solution was optimized to enhance specific antibody-antigen interactions to improve sensitivity and decrease overall background. Non-specific binding caused by low quality antibodies is reduced while signal from the specific antibody-antigen complex is stabilized and enhanced. This is my go-to blocking and Ab incubation solution since it addresses the issue of detecting low abundance antigens while boosting sensitivity. AdvanBlock-Chemi can easily be used as a substitute for all blocking solutions without any other workflow modifications. If the blocking solution still doesn’t do the trick, the antibody may be the problem. Repeat the same process as listed above for nonspecific banding.
AdvanBlock-Chemi Blocking Solution
FlashBlot Transfer Buffer
Blotting Sponge Pads