Western blotting: Tips to optimize a deceptively complex process

Tatjanacrop Tatjana Bosnjak

I was having dinner with a friend yesterday, and we started talking about Western blots and how can we make some improvements. “I can’t really tell you anything more than to follow the recipe,” my friend said. This got me thinking because it’s not actually that simple. Western blot protocol looks straightforward on paper, but it’s a multistep process, going on for days, and there are many opportunities for mistakes. There is always room for improvement, whether it’s your technique or the quality/proper choice of reagents and antibodies.

One common problem with Western blots is high background signal, which often occurs as a result of an improper blocking step. Blocking of the membrane is a crucial part of any Western protocol, and failing to do so results in non-specific antibody binding—meaning the Fc region of the detection antibody basically interacts with the membrane or with the blocking buffer itself. There is ample choice of blocking buffers available. There is no wrong one or right one—it all depends on chemical properties of your protein of interest and antibody.

Blocking buffer options:

  • Nonfat dried milk: Easily available (you can get it in any supermarket), cheap, and therefore widely used. It’s usually prepared in 0.2-5% concentrations. If you are using phosphospecific antibodies, keep in mind that this blocking buffer can cause interference, as it contains phosphoproteins.
  • Purified casein: Used in 1% concentration. It has less probability of interference than dried milk, but it still has some affinity toward phosphoproteins.
  • Bovine Serum Albumin (BSA): A bit more expensive than dried milk, but used in the same concentrations (0.2-5%), and typically interacts with phosphotyrosin antibodies.
  • Fish- and plant-derived buffers: They typically don’t cross-react with animal antibodies, but can interfere with protein-antibody interactions. They are used in the 2-10% range of concentrations.
  • Detergents: Tween 20 is the most frequently used detergent. Working concentrations are 0.01-0.1%, and it’s often used in combination with other blockers. This blocker is non-permanent and it can be removed by washing. The downside of Tween 20 is that it can disrupt protein surface or protein-antibody binding.
  • Non-protein blockers: Polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), or polyvinyl alcohol (PVA) are the non-protein blockers used often in combination with other blockers and in concentrations from 0.5-2%. They easily bind to hydrophobic membranes and cause them to become non-binding and hydrophilic.

Selecting the blocking buffer is an optimization process, dependent on your system. For example, if you are using alkaline phosphatase (AP) conjugate, you want your blocking buffer to be in TBS (Tris-buffered saline) and not PBS (phosphate-buffered saline), because PBS interferes with AP activity. The ideal blocking agent for every application does not exist because every antibody-antigen pair has unique traits. Empirically testing various buffers for your system is the best way to go in determining your blocker. You can make them by yourself in the lab, or you can buy commercial products.

Blocker troubleshooting tips:

  • The most typical problem is excessive background noise and a reduced signal-to-noise ratio. This happens due to ineffective/incomplete blocking or due to antibodies binding to the proteins in the blocking buffer. Sometimes we can also be baffled by multiple non-specific bands (i.e. bands that aren’t the target protein). Possible solutions include increasing the amount/concentration of the blocking buffer, because when it’s too low there is not enough blocking buffer to cover all of the non-specific binding sites. Another option is to block the membrane at room temperature, add Tween 20 because it increases blocking efficiency, and give it a bit more than an hour. If this doesn’t work, I recommend you leave your comfort zone and try a new blocking buffer.
  • On the other hand, we can sometimes go overboard with amounts and concentration of the blocking buffer, which results in weak bands or no signal at all because we've interfered with protein-antibody interaction. A simple way out of this is to lower the concentrations and remove any detergents present, since they, too, can be responsible for this.  
  • Have you ever seen those annoying black dots on your pretty Western blots? They are certainly eyesores, but they’re not worth worrying about too much because they're easy to fix. These dots come from clumps in your blocking buffer. The easiest solution is to filter the buffer—or you can make a fresh one and add some Tween 20, which minimizes aggregation.

Finding the right blocker for your system is all about hitting that sweet spot between low background and high sensitivity. If you use insufficient amounts of the blocker, you will end up with excessive background staining and reduced signal-to-noise ratio. Conversely, if you use an unnecessarily high concentration of blocker, you will cover up antibody-antigen interactions or inhibit the marker enzyme, and yet again cause a reduction of the signal-to-noise ratio. As I mentioned, there is no universally perfect blocker—you just need to find your match. But, as with dating, we all know how hard that can be sometimes. Good luck!

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Tatjana Bosnjak

Tatjana Bosnjak

Tatjana is a PhD Candidate at the Department of Pharmaceutical Biosciences, University of Oslo, Norway. She is interested in how different drugs and substances affect our body, i.e. pharmacology. She studies proteolytic enzymes with a particular focus on cysteine ​​proteases (legumain, cathepsins) and what roles these enzymes have in normal physiology and in diseases such as cancer, atherosclerosis and osteoporosis. Currently, her main goal is to illuminate the cellular and molecular mechanisms of legumain and cysteine ​​cathepsins in bone remodeling and bone cell functions under physiological and pathological conditions of bone loss and to utilize them as potential pharmacological target molecules.