To ensure a consistent biomagnetic separation process, all the magnetic beads should experience the same conditions. Controlling the magnetic force is key to achieving consistency within and between batches, especially when scaling up. Classical magnetic separators generate a magnetic force that is very high on the side of the vessel closest to the magnet but declines rapidly with distance. The magnetic force experienced by beads in the retention area is therefore greatest, while the beads farthest away experience the lowest force.
Proteins are one of the four macromolecule building blocks of life. The other three are carbohydrates, lipids, and nucleic acids. Proteins are long strings of amino acids that fold together into what are called “hierarchical structures” in order to perform specialized functions within the cells and tissues of all living organisms.
Customizable Nanoframeworks are one of the most exciting innovations in the world of nanochemistry. There are two main classifications of nanoframeworks. The first is the Metal-Organic framework (MOF). A MOG is a classification of a compound that consists of a metal linked to an organic ligand to form a coordinated structure in 1, 2 or 3 dimensions.
The second is a Covalent-Organic framework (COF), which is a crystalline porous organic framework with two or three dimensional properties. A COF is usually, but not always, limited to light elements (H, B, C, N and O) . Both possess a π-conjugated system and have a wide porous volume that can be tuned with the selection of a linker. This linker also has further effects on the electronic structure of the material. Thousands upon thousands of different, unique frameworks have been identified, leading to a variety of sizes that range from the nm to mm range. However, in all cases, the porosity of the framework benefits from a high surface area to volume ratio, leading to many different applications using a delivery mechanism that benefits from rapid diffusion.
Sepmag starts distributing its unique Biomagnetic separation systems in India, where Biotechnology industry is fast developing. In Vitro Diagnostic, protein purification and cell sorting companies working with magnetic beads will benefit enormously from the quality, reliability and monitoring that Sepmag technology offers to biotech enterprises around the globe.
Traditionally, biomagnetic separation users have not monitored the separation process. The nature of classical separators, where the magnetic force changes with the distance, means you can determine when the separation is complete (the buffer becomes transparent), but it is difficult to interpret the optical changes during the process. This is because every location sampled will have a different bead concentration due to their different speeds. In addition, it is difficult to compare different batches as even a small difference in the vessel’s position within the separator will affect the beads’ behavior.
Batch adsorption with magnetic beads is considered a mild technique, which is advantageous for the purification of sensitive plasma proteins. Cytiva’s patent application shows excellent results using magnetic beads with suitable ligands for the purification of Factor VIII and von Willebrand Factor in solubilized cryoprecipitate, and Factor IX in cold supernatant. The magnetic separation system, with constant magnetic force, used in these experiments enables the straightforward transferring of the protocols to large-scale volumes.
The Importance of protein purification buffer
A buffer, by definition, resists changes in pH when small quantities of acid or base are added to it. Protein purification protocols utilize several types of buffers to aid proteins in your solution in binding to your separation mechanism, then washing out unnecessary molecules from the solution, and finally to elute the purified protein and store it. There are many types of buffers that come at different pH’s. Depending on need, scientists will use additives such as salt, as well as protease inhibitors to create the ideal protein purification buffer for their protein. Common Buffers are Tris-HCl, HEPES-NaOH, MOPS, etc. At the low end of the pH range, citric acid-NaOH can be used in the 2.2 to 6.5 pH range. MES-NaOH is closer to pH 6, while imidazole-HCl is around 7. Tris-HCl is up around pH 8 while HEPES-NaOH is between 7 and 8. Differences in pKa, the strength of the buffer, can arise from differences in temperature of the buffer. After using an elution buffer to elute your protein, you will want to quickly neutralize it with a storage buffer to keep the protein from experiencing damage.
Immunoaffinity chromatography is a method for separating target antibodies or antigens from a heterogeneous solution. It is column-based, which means that the solution is flowed through a column and eluted at the other end. The column is pre-functionalized with the capture antibody or antigen. The target protein is adsorbed onto the resin-bound capture protein and is retained in the column while the remaining solution is eluted. The fraction containing the target protein is later eluted and purified.
Cube Biotech’s video explores magnetic beads as a solution for large-scale protein purification using a Sepmag A400ml.