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Posted on Thu, Jan 18, 2024

Protein Purification Buffer

The Importance of protein purification buffer

A buffer, by definition, helps a solution to resist changes in pH when small quantities of acid or base are added to it. Protein purification protocols utilize various buffers to aid in stabilization at each step of the process. For example, lysis buffers are initially used to establish an ionic strength in the solution, and binding buffers help in target proteins bind to an immobilizing support matrix, like magnetic beads.

After the sample is washed, an elution buffer is used to disrupt the interaction between affinity ligands and the target molecule. Key attributes of protein purification buffers are that they must prevent protein unfolding and aggregation, while simultaneously not interacting with the target protein. Common protein purification buffers are Tris-HCl, HEPES-NaOH, MOPS, BIS-TRIS, PIPES, and TES. There are many types of protein purification buffers that come available in different pH’s, in a range of molarities, and with various salt concentrations. Reducing agents, stabilizing elements, and additives can also be included into your buffer to increase the solubility and stability of the protein complex.

Free PDF guide:  "Basic Guide to Recombinant Protein Purification" 

Key elements in a protein purification buffer

In determining appropriate protein purification buffers, you must first consider the method of purification, the cell samples to be analyzed, and the intended results of the experiment. In determining an optimal buffer, first identify a suitable pH range;. If the protein purification buffer is too close to the isoelectric point of the target protein, then the protein will not be able to bind to the support matrix. Next, it is important to choose a buffer with a pKa value within approximately one pH unit of your desired pH. The pKa value is often viewed at the strength of an acid or base, though this value is also dependent upon buffer concentration and temperature.

Generally protein purification buffers also include salts to help keep proteins soluble and also mimic physiological conditions. Salts, like NaCl, may be added at slightly more or less than 100mM to maintain the ionic strength of the solution. Additionally, reducing agents can be included to reduce di-sulfide bridges that may become oxidized on cysteine amino acids in proteins. Common reducing agents are used between 1-10mM, and include DTT, beta-mercaptoethanol, and TCEP. Detergents, like dodecyl sulfate (SDS) or Triton X, can also be incorporated into a protein purification buffer to stabilize membrane protein in solution for the purification process. Lastly, there are a number of additives that can help increase the accuracy and effectiveness of a protein purification experiment. Glycerol, PEG, and BSA have all been documented to prevent aggregation and stabilize proteins while some ionic compounds like sulfates, amino acids, and citrate have shown  to help shield ionic interactions and solubilize proteins.

Magnetic protein purification buffers

There are still a few things to keep in mind in determining appropriate buffer conditions for magnetic bead protein purification. Due to the high throughput capability of modern magnetic bead techniques, the initial steps in protein purification may require large volumes (think liters worth) of magnetic protein purification buffer. When preparing washing buffers after the isolation of the target protein, non-ionic detergents may be added to help minimize non-specific binding by the magnetic beads. Elution buffers used in magnetic bead protein purification also usually have a high salt concentration or induce a change in pH to elute bound DNA/RNA binding proteins. Importantly, only very small volumes of magnetic protein purification buffer is required at this step, because just milliliter volumes of protein are eluted.

There is no doubt that advances in magnetic bead technology have increased the scalability, stability, and efficiency of protein purification techniques. With more and more research advancing magnetic bead technologies, optimization of magnetic protein purification buffers has become increasingly simplified. Though many buffers are commercially available, protocols for building in-house buffers are also widely available in the literature. Key ingredients and additives are also widely available, and should be considered so increase the durability and performance of a protein purification experiment.

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Published on November 26, 2020 and updated on January 18, 2024.

 

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