With the advent of pharmaceutical biotechnologies in recent years proteins and peptides have been the main focus of numerous studies by researchers and companies. Peptide and proteins have various physiological functions in body (as hormones, enzyme substrates and inhibitors, biological regulators, structural components, signaling factors, catalyzers), peptide/protein-based drugs and biopharmaceuticals are a novel category of drugs, and any abnormality in their amino acid sequence or structural dysfunction can lead to severe diseases and pathological conditions (dwarfism, cystic fibrosis, thalassemia etc).
Proteomics is the study of the protein in an organism. Protein is a fundamental building block of life, and proteins are the workhorses within and between cells. Biochemical pathways are built out of enzymes and ligands—without them nothing would be accomplished; plants wouldn’t produce glucose, animals wouldn’t be able to digest food, the immune system would cease to exist, and all other biological processes would grind to a halt. The fundamental importance of proteins for life makes them an important topic of study. The first step in understanding protein structure and function is to extract them. Protein extraction is the process of isolating and purifying protein from samples of whole tissue, cell cultures, or biological fluids. The protein extraction protocol used is tailored to match the starting material and the end goals of the assay.
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.
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.