When it comes to biotechnology research, protein engineering techniques follow. In other words, different protein engineering techniques are used. The goal is often to design or modify immunoassay protein. Remember, this is done with a specific application or aim that relates to the biotech industry.
Scientists will apply a specific technique to isolate a specific strain of protein before it undergoes the purification process. This is done so that scientists can learn, understand, or study the conformation and specification of the substrate through research. Furthermore, the reaction between protein and other ligands is conducted; not forgetting enzyme-specific activities.
Ligand is defined as a protein that attaches to a special receptor.
During protein purification, the level of purification generally depends on the demand. For example, in some cases, a simple crude extraction is all that is needed. When it comes to some fields like the food or pharmaceutical industry, high purification levels are at-most important. For this reason, such industries apply different but appropriate purification techniques to achieve the desired results.
It is wise to note that each protein purification process or technique reduces product content. For this reason, the best and ideal purification techniques are those with few steps but offer high purification levels. Nonetheless, there are several factors that will influence the steps or process of protein purification. Some of these factors include:
• Protein size
• Protein Charge
• Protein Solubility and much more
Below is a procedure that is best for purifying single cytosolic proteins. Remember, in case you are working with cytosolic protein complexes, you will need different techniques.
1. Crude Extract Preparation
Crude extract preparation is the first major step in intracellular protein purification. Intercellular proteins are defined as protein types that are found inside a cell. The crude extract contains a complex mixture of protein, nutrients, cofactors, and macromolecules, which has several biotechnology applications.
However, after preparation, if purification becomes a problem, there are several steps that a scientist must undertake. This can be accomplished by using chemicals, enzymes, sonication, or a French Press.
2. Crude Extract Debris Removal
For a scientist to remove debris from the crude extract, a centrifugation process must be used. The supernatant can be removed successfully after the completion of the process. When it comes to extracellular preparation, the protein is generally obtained after the completion of the centrifugation process.
Thermostable enzymes are required in some biotechnology practices. It is important to note that thermostable enzymes are those that can withstand high-temperature levels. Furthermore, they maintain a high level of activity.
3. Intermediate Protein Purification Steps
Today, a majority of modern biotechnical protocols have taken advantage of kits that are available at a commercial level. Additionally, they apply techniques that offer ready-made solutions for standard procedures. For instance, filters and gel-filtration columns are widely used when undertaking protein purification activities.
4. Dialysis Kit
It is wise to always follow the directions or instructions provided by the kit. These directions act as a guide to ensure the correct amount of solution is used while following the appropriate waiting time. These kits specify the waiting time for the right concentration of fluid to enter the clean test tube. In a nutshell, it is very important to follow the directions given with any dialysis kit.
5. Chromatographic Methods
This method can be achieved with either a bench-top column or automated HPLC equipment. With HPLC, size exclusion, reverse-phase, or ion exchange method must be used. Obtained samples are detected using a diode array or laser technology.