Introduction to RNA extraction kits
Why do we need immunoassays kits?
There are many types of Immunoassay kits that are used in laboratories, clinical settings, and in industry. A typical laboratory bench-top kit is the ELISA kit, which stands for enzyme-linked immunosorbent assay. In a core facility or clinical setting you will find larger instrumententation for assays such as the latex turbidity immunoassay (LTIA) or chemiluminescent immunoassays (CLIA).
The Importance of protein purification buffer
Protein purification protocols call for 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.
Fundamental research often involves the study of isolated cell populations. It is these enriched populations that enable researchers to make new discoveries about cell function, signaling, gene expression, fate decisions, and much more. Techniques for the rapid and accurate enrichment of target cell populations are an area of great interest. Cell sorting techniques fall into two general categories: bulk sorting and single cell sorting. In bulk cell sorting all of the target cells are collected in one sweep, whereas in single cell sorting every cell is individually analyzed. There are multiple methods of bulk cell sorting: filtration, centrifugation, and magnetic cell sorting. The main single cell sorting method is flow cytometry or fluorescence activated cell sorting. While cell sorting can be very accurate, it is hard to say that a sorted cell population is “pure”. Instead, the collected population is referred to as “enriched”. In general, single cell sorting results in highly enriched cell populations that are more homogeneous than those obtained via bulk sorting methods.
A couple of months ago we described the sandwich elisa. Here we will discuss the other two main types of elisas—indirect and direct. Elisa is an acronym for enzyme-linked immunosorbant assay. The purpose of any elisa is to detect the presence of an antigen or antibody of interest. The indirect and direct elisa differ from the sandwich elisa because the antigen of interest is bound directly to the plate rather than a capture antibody. In either case, the key component is an enzyme-linked detection antibody. The enzyme is either colorimetric or chemiluminiscent. Chemiluminiscent enzymes are popular because they are easily read by a luminometer plate reader, making the process easy and highly quantitative.
Overview of immunoprecipitation with magnetic beads
Immunoprecipitation is a technique that uses antibodies to purify a molecule of interest out of solution. In a general protocol, an antibody against your molecule of interest will be pre-bound to a magnetic bead. The pre-conjugated beads will be mixed with a mixture, such as a cell lysate, and antibodies will bind their specific target in solution. When the mixture with beads is placed in the presence of a magnetic force, using a classical magnetic separator or modern biomagnetic separation system, the beads will be tightly held against the side of their container. Modern magnetic separators have been innovated to provide many sizes of magnets and they overcome challenges with the strength of the force for efficient separation. Modern separators provide a constant force that allows for stable separation over time. With the beads so strongly immobilized, one can simply remove the liquid from the container and replace it with a new clean buffer. When the container is taken out of the magnetic field, the beads will go into solution, still bound to your molecule of interest, now precipitated out of their original solution. 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.
The magnetic cell separation technique: Executing cell separation by magnetic activation has been a trusted technique by scientists for decades. Cell sorting is ubiquitously used in research and clinical settings where a target cell of interest needs to be isolated from a heterogenous mixture such as serum or plasma. It is used in several scientific disciplines such as immunology, where it helps identify cells present during immune responses, or in cancer research elucidating tissue environment of tumors.
Microbeads are composites of nanosized magnetic particles embedded in a non-magnetic matrix, the size of each bead in the range of micrometer diameter. Microbeads were originally the discovery of John Ugelstad, who was able to create uniform polystyrene spheres. Magnetic microbeads are superparamagnetic microbeads that specifically only become magnetized in the presence of a magnetic field, and this is a reversible phenomenon. These microbeads, made of materials with magnetic properties such as magnetite, have several uses in scientific research. An important use of magnetic microbeads is for separation of biological molecules from mixtures.
Magnetic bead technology can be used to separate several types of biological molecules from the solution which they are in. You can separate a protein of interest or a nucleic acid, DNA or RNA, from a heterogeneous mixture such as cell lysate. There are kits and protocols that have been established and are commercially available for isolating DNA or RNA from a solution. These kits contain buffers that can make the isolation process time efficient, and keep the yield of nucleic acid recovery high. Beyond that, some laboratories are investigating an optimization for isolating both DNA and RNA with a protocol that is even more time and cost effective.