Buffers are perhaps the most widely used solutions in a lab. They’re mainly used for controlling shifts in pH, which is crucial for any biochemical system. However, there’s more to buffers than this.
In 1966, Norman Good set out to define the best buffers for biochemical experiments and established several criteria to consider when choosing the right ones to use. More than 50 years later, his buffer selection criteria still applies.
So, how can you choose the right buffer for your proteins?
Determine the optimal pH for your experiment
pH influences the chemistry of amino acids and can therefore greatly influence protein structure and function. Even small changes in pH can lead to protein unfolding, aggregation, functional inactivity and even affect molecular interactions including protein-protein and protein-ligand. Therefore, in order to keep your proteins “happy” while preventing these unwanted effects, the first thing to do is to determine at which pH you need to work. For example, if you’re working on an enzyme assay, choose a pH where your enzyme works at its maximum activity. Or if you’re planning an ion exchange purification, choose the right pH to have your protein charged as it’s needed.
Focus on buffering capacity
Once you know the pH range you’ll be working at, it’s time to start choosing a buffer. But before doing so, keep in mind that each buffer has a range of pH in which they are effective, meaning their buffering capacity. To choose the right buffer for a selected pH, a rule of thumb is to pick a buffer with a pKa value within one pH unit of your test. This will ensure that your experiment’s pH will remain in the desired range, keeping your proteins safe and sound while preventing unwanted changes in their behavior.
Note that the buffer pH varies in function of temperature, therefore you need to check pH at the temperature planned for your experiment.
Assess protein stability under different buffer conditions
Buffers can alter protein structure, function and even the rate of aggregation with either favorable or adverse outcomes depending on the protein. Therefore, while a particular buffer may protect a protein against freezing damage or functional inactivation, it may promote the aggregation or degradation on others. This is why screening protein stability in the presence of your candidate buffers is an extremely important task because it can lead to the perfect buffer for your proteins and greatly simplify the interpretation of your results.
To understand the impact of a buffer on the stability of your protein, you can screen with Prometheus, which can characterize thermal unfolding, chemical denaturation and aggregation in a single run.
So remember, buffers can have profound effects on protein stability and therefore have a major impact on your experimental results than you think.