Protein biochemistry brings together a vast and varied world of methods of protein production, purification and characterization. Once you have successfully achieved the production of your protein in a selected system, you need to think about the following steps. Indeed, the quest does not stop there! The next step is purification, during which you will try to isolate your protein of interest from the surrounding contaminants while keeping it soluble and active. Your ultimate aim is to keep your protein “happy” by choosing the perfect buffer. You will be faced with a bewildering array of choices leading you to ask yourself “where do I start…?”.
Keep calm – it’s easy…
I’d like to offer you this simple guide, which will allow you to easily select the right basic buffer for the right protein purification strategy in order to facilitate your decision making. Obviously, it is not so simple and it will require some optimization, but I think that a “what-to-do-first-guide” is always welcome!
First of all, a buffer solution is one which resists changes in pH when small quantities of an acid or an alkali are added to it.
A good buffer needs to have some specific characteristics like:
- Solubility in water
- Chemical stability
- Highly efficient buffer at a chosen pH
- Compatibility with other components of the buffer
- Compatibility with subsequent experiments
2# Context evaluation
The first thing to do is to determine at which pH you need to work. For example, if you’re planning an ion exchange purification you need to choose the right pH to have your protein charged as you want. Indeed, when the pH buffer is equal to the pI (isoelectric point), the protein has no charge. Consequently, the protein is not able to bind to an ion exchange column (anion and cation). Adjusting the pH below or above the protein pI induces a positive charge when pH < pI and a negative charge when pH > pI (Fig.1).
When the protein has the complementary charge of the resin, the protein is able to bind to the column and the elution can be enhanced by varying the pH or by increasing the salt concentration.
Another example is the hydrophobic interaction chromatography, also named HIC, by using a resin containing hydrophobic groups. Proteins in solution have various surface residues which can be hydrophilic or hydrophobic. The resin hydrophobic groups are able to bind proteins from aqueous solutions to different extents depending on the buffer components. The most important thing is to have a good binding selectivity. The interaction is enhanced by buffers with high ionic strength (high salt concentration), which makes HIC an excellent purification step after elution in high salt during ion exchange chromatography (IEX) for example. This interaction is reversible. For selective elution, also named desorption, the salt concentration is lowered gradually and the sample components elute in order of hydrophobicity.
To summarize, each purification strategy has its buffer recommendations. First, you need to choose the purification strategy and after that you can select the most adapted buffer.
3# Selection of buffer Components
Each buffer has a range of pH in which its buffered power is efficient. To be sure that you have chosen the right buffer for a selected pH, you can take a look at Tab. 1 which summarizes the commonly used buffers and their range of use.
For example, if you need to have a pH around pH 7, PIPES, HEPES and MOBS can be used because of their buffering capabilities at this pH. It’s very important to choose a buffer that has a pKa value within one pH unit to your desired pH. Commonly, a concentration between 25-100 mM can be used but you need to be sure that this component concentration is able to efficiently buffer the solution.
Note that the buffer pH varies in function of temperature and that you need to check pH at the temperature planned during purification.
A purification buffer is also composed of several components that are named additives, which can play an important role in protein quality. These additives can be grouped into several types of additives with different functions (see Tab. 2). For example, DTT (Dithiothreiotol) is a reducing agent which protects protein against oxidation and related damages. Some of them are able to inhibit protein degradation by endogenous proteasis like PMSF, a protease inhibitor. Others, classified as Osmolytes, have the ability to stabilize the protein structure and to enhance protein solubility (eg. glycerol, detergents and sugars). Some ionic stabilizers like salts enhance the protein solubility. The protein folding can also be promoted by alpha-helix stabilizers which include TFE, TMAO and chaperon proteins.
The ultimate aim of protein purification is to conserve a large quantity of functional protein with less contaminants as possible. Determining the best buffer for a protein of interest requires some development. The information in the tables above can guide you in selecting a buffer screening adapted to your case and to keep your protein “Happy”. Enjoy your testing!
For queries or help, don’t hesitate to contact us – or leave your questions and comments below!
Hi, I would like to know how to stablish the optimal conditions for a protein, maximizing solubility. How can I design an experiment to evaluate pH? What buffer may I chose?