Introduction to RNA extraction kits
Immunoaffinity chromatography is a method for separating target antibodies or antigens from a heterogenous 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.
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.