Protein extraction is a key step for many proteomics research procedures, from ELISA to Western Blot. Proteins form the basis of all cells, tissue, and organisms. Proteins also initiate and mediate the thousands of biochemical pathways that govern an organism’s function. Biomedical studies of proteins can reveal information about pathways of disease, and the expression of the genetic code. But before proteins can be studied, they need to be extracted. Choosing the most appropriate protein extraction method is key to successful protein extraction.
The earliest chemists were on the hunt for new elements to add to the periodic table. Most of the chemistry that they were interested in doing was purification with the end goal of reaching a pure elemental substance. These chemists relied on a litany of methods—filtration, evaporation, distillation, and crystallization were some of the most used purification techniques for these discoveries. As the chemists were defining the elements, the biologists were trying to understand the human body, the cell, cellular organelles, and microbes. The point here is that in order to develop anything new we must first understand what everything is made of at the most basic and pure level. In modern science this means that we are trying to define matter beyond subatomic particles and we are attempting to map out every molecular pathway of disease. Our efforts to define complex systems by their purest constituents are rewarded by deep understanding and an ability to mimic, to engineer, develop, and create.
Protein purification is the processes of isolating a protein of interest from its environment. In other words, from the other natural molecules surrounding the proteins in the natural niche in a host organism, or from a cell culture grown in a laboratory. Our protein purification handbook explains that there are several available techniques and many options to consider, but the general procedure is the same.
Most current protein purification methods use agarose beads carrying affinity functionalities such as IMAC, Glutathione, or antibodies. The choice of these functional groups depends on the protein of interest to be purified, and a large variety is available, including pre-functionalized beads that can be coupled to biomolecules (see SEPMAG® protein purification handbook chapter 4 and 5).
Magnetic particle imaging (MPI) is a new technology that uses the signal generated by superparamagnetic tracers generated by changing magnetic fields. As it is not a natural superparamagnetic substance in the human tissues, the resultant images have no background. The tracers used in magnetic particle imaging are superparamagnetic iron oxide nanoparticles (SPIONs). The optimization of magnetic nanoparticles (MNP) plays an essential role to improve the image resolution and sensitivity of imaging techniques.
Antibodies are a key component to many biotechnical applications. They are most often used for immunoassays such as ELISA, cell and tissue staining, protein quantification such as western blot, and cutting edge sensor development. Verified antibodies are easily purchased from commercial vendors. These antibodies can be monoclonal or polyclonal, and can come as a lyophilized powder or as a premixed solution. All of these details must be considered when choosing which antibody to purchase because they all have an effect on the antibody concentration and dilution process.
Chromatography systems, or purification systems can be used to purify protein, nucleic acids, or just peptides. It comes in different sizes for different scales of purification. Research labs often do purification in smaller batches and in industry settings companies do large scale purifications. The AKTA pure is an example of one such useful technology for automating the purification process, avoiding human errors, keeping the purification at a regulated temperature such as if you put the machine in a colder environment for less stable molecules, and having a consistent and regulated amount of pressure applied to purification columns.
mRNA purification: how mRNA vaccines work
The letters “mRNA” are heard everywhere lately. The mRNA vaccine has been widely distributed in response to COVID-19. The mRNA in the vaccine enters cells in the body where the cell machinery can translate the mRNA into the Spike surface protein of the coronavirus. The body recognizes the spike protein as an antigen and produces antibodies against it. When infected with the actual virus later on, the immune system has a base defense system, antibodies, ready to more specifically remove virus.
An antigen is defined as anything that causes an immune response in another organism. This immune response can be a simple increase of inflammatory factors, or it can be an activation of the adaptive immune system and creation of antibodies. Antibodies have two or more specific paratopes, or antigen recognition sites, that identify and combat the invading antigen. The number of antigen recognition sites is dependent on the antibody class. The word “antigen” can also refer to any protein of interest detected by a bioassay or biodetection platform. In the case of a bacterial antigen, we are referring to surface proteins, lipopolysaccharides, and peptidoglycans on the bacterial cell wall; these structures help bacteria invade other organisms by gaining access between epithelial cells. While surface structures help bacteria infect other organisms, they are also a detriment to the bacteria because they also serve as a unique tag that antibodies and bacteriophages can recognize. Bacteriophages are viruses that attack bacteria. Both antibodies and phages are being used by scientists to develop new biodetection and biosensing platforms for rapid detection of bacterial antigens in the environment and in clinical samples.
Cell lysis is the act of breaking the cell membrane to enable the study of specific proteins, nucleic acids, and other molecules inside of cells. When cell lysis is successful, the undamaged contents of the cell escape through the damaged cell membrane. These contents are then separated out of the mixed sample and used for further study. The methods used for separation of the lysed cell contents are dependent on the goal of the study. Careful investigation of these inner workings can reveal disease patterns, improve our understanding of normal cellular function, and elucidate biochemical pathways and therapeutic targets. Protein isolation is different from nucleic acid separation, and the reagents used vary drastically. There are a few ways to lyse the cell membrane; these include mechanical disruption, liquid homogenization, freeze/thaw cycles, manual griding, and the use of detergents. Sonication cell lysis is an example of mechanical disruption used for releasing the contents of cells.