The separation of charged biomolecules of a solution by their size and molecular weight is common for scientific studies and biomedical techniques for which various techniques of “electrophoresis” use an electric field, protein electrophoresis is among the widely used procedures.
Protein A
Protein A is a protein derived from the cell wall of the bacteria “Staphylococcus aureus” and can also be extracted from the culture supernatant. It has unique binding properties to the Fc and Fab region of IgG of some mammalian species (and to a lesser extent to IgM and IgA as well).
Poly-L-lysine is a synthetic amino acid polymer – a chain ofmultiple L-lysine amino acid monomers, with multiple industry and biomedical applications.
As a monomer, lysine contains two amino groups, called α-polylysine and ε-polylysine, based on which carbon they are on. Α-polylysine is composed of either L-lysine or D-lysine enantiomers, based on the chirality (“handedness”) of the lysine’s central carbon. Poly-L-lysine is a polypeptide formed from L-lysine monomers. Poly-D-lysine, a similar molecule used in similar applications, is a polymer formed with D-lysine units.
Principles of immunoprecipitation with magnetic beads
Immunoprecipitation is one of the commonly used antibody-based techniques which relies on the specific affinity of an antibody to identify its target molecule within a biological sample for further analysis and research.
In a general protocol, antibody is pre-bound to a scaffold (magnetic beads or agarose beads) while it is incubated with lysates containing the target molecule. The mixture is then placed in the presence of a magnetic force. Using a classical magnetic rack the beads will be tightly held against the side of their container. The buffer simply can be removed from the container while the target molecule is still bound to the strongly immobilized beads to be finally eluted. This can also be done with free antibodies, which can be mixed with a cell lysate to bind their target, and then are bound to magnetic beads.
Magnetic separation is rapidly gaining popularity in the field of Life Sciences due to its numerous advantages. When considering the adoption of magnetic separation to purify biomaterial, it is crucial to recognize that the magnet itself plays an equally vital role as magnetic beads in achieving efficient biomagnetic separation.
Magnetic bead-based fluorescent immunoassays can detect and measure single or multiple analytes, such as certain proteins, present in one sample. The technology uses fluorescent magnetic beads, such as StrepTalontm or Luminex® beads, and detection antibodies to detect multiple analytes, and therefore answer multiple questions, simultaneously. So how do they work?
Importance and uses of cell isolation
Cells are the basic and fundamental units of organisms carrying genes for the biological function of that particular cell. Higher organisms (eukaryotes) are composed of 200 different types of cells (e.g. red blood cells, white blood cells, muscle cells, bone cells, nerve cells etc.) that have different gene expression profiles and even the same cell lines can present different genomes, transcriptomes and epigenomes during cell division and differentiation.
Transfection is a technique that makes it possible to modify the genetic content and therefore gene expression of host eukaryotic cells, both in vivo and in vitro. This makes transfection an important tool for drug discovery, the CRISPR-Cas9 technique, studying cell biology, cellular functions and molecular mechanisms of disease. The process centers on inserting proteins, nanoparticles or nucleic acids (such as cDNA, mRNA) into the cytoplasm of cells.
Previously, the use of magnetic beads was limited to small volumes. The difficulty of scaling up beyond a few milliliters was misinterpreted as a limitation of the technology itself. However, as discussed in this e-book, the problem is not the biomagnetic separation process, but a lack of understanding of the physical processes governing it. Once you identify the key parameters that control the magnetic bead's behavior, it is easy to choose the right tools and methods to validate the process and replicate it at different volumes.
Immunoassay tests are biochemical/bioanalytical methods that detect an “analyte” and quantify its concentration in a complex mixture of chemicals or biological fluids (e.g. serum or urine). Analytes can be a micro- or macromolecule (e.g. protein, nucleic acid, polysaccharide or lipid) or chemical substances (e.g. hormones, drugs).
