The selection of buffer fluid in protein preparation and purification process is very important, and suitable buffer fluid can ensure the activity and structural stability of protein.
An overview of the
When we purify the protein, the most important thing is to keep the protein not inactivated during the purification process, or to minimize the loss of the purification process to the protein activity.
This means that the protein remains soluble and active throughout the purification process.
Therefore, it is very important to design a protein purification buffer to prevent protein degradation and polymerization, especially in the experimental results.
The following factors should be considered when designing buffer buffer: pH value, buffer system, salt ion, reducing agent and stabilizer.
Each of these factors is optimized according to your target protein, and the activity of the target protein is used as the optimal evaluation criteria.
1. The pH
Many experiments have a pH value of 7.4 to mimic biological conditions.
If your protein is stable at this pH, that's great!
If it is not, you need to change the pH to find the target protein in the solution in a soluble and non-degradable state.
A rule of thumb is: protein in its isoelectric point near pI will behave in the pH of the solution is not easy to dissolve, because protein in its isoelectric point no net charge in the pH of the solution surface, thus easy to gather.
It is recommended to use the ProtParam tool to quickly and easily calculate the point pI value of the protein, as long as the protein sequence is submitted.
2. Buffer system
Once you determine the pH of the buffer, you need to choose which buffers to use.
The first thing to do is to make sure that the buffer system you choose to have a buffer in your buffer system is really buffering.
The buffer system of choice, the solution of the dissociation constant pKa value should be within a pH value unit of the set pH.
Also, make sure that the buffer concentration you use is high enough to reach the actual buffer solution, and the normal concentration is 20 ~ 100mM.
It is important to remember that the buffer system you use should not affect the activity of the protein!
For example, phosphate can inhibit kinase activity, so it should be dialysis thoroughly before the reaction.
In addition, some buffer system is very sensitive to temperature, Tris - HCl buffer, for example, if your buffer system at 25 ℃ to pH 8, pH value will increase to 8.58 at 5 ℃ and at 37 ℃ to 7.71.
So, if you plan to in the 4 ℃ under the condition of preservation proteins or 37 ℃ in the experiment, you should consider to room temperature lower pH may not applicable in the experimental conditions.
3. The salt ions
Many buffers contain NaCl to help maintain the protein's soluble and simulated physiological conditions.
Generally used, 150 mM NaCl.
In different protein purification steps, the concentration of salt ions may need to be changed.
If, for example, is a protein purification by ion exchange chromatography, first need to reduce the concentration of the salt ions (5 ~ 25 mm) to avoid high ionic strength and protein competition and packing, so can prevent the protein was wearing from ion exchange column, so that the protein pillars can be combined with purpose, elution to remove impurity protein.
Other types of chromatographic separation columns, such as gel filter columns and Ni2 + affinity chromatography columns, may require higher salt concentrations.
I usually use up to 500 mM NaCl for high salt leaching to prevent non-specific binding between protein and column.
Finally, the target protein was replaced by a gel column in the system of a new buffer concentration.
4. The reducing agent
If your protein contains cysteine residues, the oxidation of cysteine residues may become a trouble, and may lead to protein aggregation.
To prevent this, some reducing agents, such as DTT, TCEP or mercaptoethanol, are added to the buffer.
Overall, TCEP is the most stable of the three reducers, but it is also the most expensive.
I usually add DTT in the buffer of the purification process to add TCEP to the buffer of the last preservative.
Generally reasonable reducing agent concentration is 5 ~ 10mM.
Basically you want to make sure that the reductant concentration is much higher than your protein concentration.
Because DTT and mercaptoethanol are degraded at room temperature, the buffer that has been added to the reducer is required to be kept at a low temperature, or added to the reducer when used.
Use the material to ensure that the column material can be compatible with these reductants.
For example, a high concentration of reducing agent will take off in nickel, nickel column and post darker brown color, can choose high resistance nickel column at this time, although pillar is very easy to regenerate, but your protein hanging loads will be badly affected.
Many of the column materials will list the maximum concentration of the reductant, but I think more or less will have an impact, not whether the maximum concentration is achieved.
5. Stabilizer
Finally, the solubility and stability of the protein purification can be improved by adding some stabilizer to the protein purification buffer.
The addition of inert protein BSA to the buffer may stabilize the protein, but it is important to ensure that these added stable proteins do not interfere with the experiment.
Sometimes additives such as glycerol and polyethylene glycol can increase the viscosity of the buffer, which can help prevent protein accumulation.
In addition, using a small amount of surfactant and some ionic compounds such as sulfates, amino acids, citric acid and other proteins can block the interaction of the ions, helping to dissolve the protein.
By adjusting the pH of protein purification buffer, buffer system, salt ions, reducing agent and stabilizer, can build a perfect protein purification buffer system, can maintain protein activity and stability in the purification process.
This production adjustment is very beneficial to the production process development of protein products, enzyme preparation products and antibody drug products separation and purification.