When purifying a protein, it’s important to keep your protein happy. If you are going to use the protein in binding and activity assays, such as the surface plasmon resonance (SPR) technique, then your protein needs to be soluble and active. For success in these experiments, it is crucial that you create a buffer that prevents unfolding and aggregation. To design the buffer, you should consider the following factors:
As described below, each of these needs to be optimized for your protein of interest.
Many experiments are done at pH 7.4 to mimic biological conditions. If your protein is stable at this pH – great! If not, you need to change the pH to find conditions that keep your protein in solution. One rule of thumb is that proteins are generally less soluble at their pI value, which is the pH at which the protein has no net charge. A quick and easy way to calculate a protein’s pI from its sequence is to use ExPASy’s ProtParam tool.
Once you’ve decided on a pH value, you need to decide which buffer you are going to use. The most important thing to keep in mind when choosing a buffer is to make sure that your buffer of choice has buffering capabilities at your pH of choice. Choose a buffer that has a pKa value within one pH unit of your desired pH.
The second most important thing is to ensure that the concentration of buffer you are using is high enough to buffer the solution. Concentrations between 50-100 mM are common.
Keep in mind that the buffer you use should not interfere with the activity of your protein—this is especially important for SPR techniques. For example, phosphate inhibits kinases and should be thoroughly dialyzed out before performing reactions. Also, some buffers are sensitive to temperature. Tris is notorious for this. For example, if you pH your buffer to pH 8.0 at 25°C, the pH will increase to 8.58 at 5°C and decrease to 7.71 at 37°C. So, if you plan to store your protein at 4°C or do your experiment at 37°C, take into consideration that the pH you measured at room temperature may be different under your experimental conditions.
Many buffers contain NaCl to help keep proteins soluble and to mimic physiological conditions. Generally, 150 mM NaCl is used. However, during various protein purification steps, you may want to change the salt concentration. For example, if you are purifying your protein by ion exchange chromatography, you want to start with a low concentration of salt (5-25 mM). This will help screen ionic interactions and prevent nonspecific binding of proteins to the column while enabling your protein of interest to bind the column. In other types of chromatographic separations, like gel filtration and Ni2+ affinity columns, you may want to increase the salt concentration. I’ve gone up to 500 mM NaCl to prevent nonspecific interactions between proteins and the column.
Expert tip: Change the salt concentration by dialyzing your protein into a new buffer.
If your protein contains cysteine residues, oxidation could become a problem and cause protein aggregation. To prevent this, keep a reducing agent such as DTT, TCEP, or 2-mercaptoethanol in your buffer. In general, TCEP is the most stable of the three, but it can be rather expensive. I often use DTT in my buffer during purification and then add TCEP to the final buffer. A good concentration to use for these reducing agents is between 5-10 mM. Basically, you want to make sure that the concentration of the reducing agent is well above your protein concentration.
Because DTT and BME break down at room temperature, keep these buffers in the refrigerator. Alternatively, make the buffers without reducing agent and add the reducing agent when you’re ready to use the buffer.
Make sure any resins you use are compatible with reducing agents. For example, high concentrations of reducing agents reduce the nickel in nickel columns and turn the column brown. The column can be regenerated, but your protein is not likely to bind well. Many columns have suggested maximum concentrations of reducing agents that the column can tolerate; however, I’ve found that this is really trial and error.
Finally, there are a whole slew of additives you can add to your buffer to help increase protein solubility and stability. You can try adding an inert protein like BSA to your buffer. This can sometimes stabilize a protein, but you must ensure that the protein you’re adding does not interfere with your experiment. Sometimes it helps to increase the viscosity by adding agents like glycerol or PEG. These typically help prevent aggregation. Also, some detergents and other ionic compounds like sulfates, amino acids, and citrate can be used in small quantities to help shield ionic interactions and solubilize proteins.
So there you have it. By keeping these five things in mind: pH, buffering system, salt, reducing agents, and stabilizing agents, you are well on your way to creating a buffer that will keep your protein happy and active for use in various analytical techniques (including SPR applications).
A former colleague and friend of mine worked in technical support, taking calls from scientists. Most of the calls came from life science researchers frustrated by failed experiments. My friend would listen for a bit, then almost always exclaim in very charming but booming Russian-accented English, “No! You must add buffer to the experiment! It […]
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