Filtration is a simple technique used to separate solid particles from suspension in a liquid solution. There are many filtration methods available, but all are based on the same general principle: a heterogenous mixture is poured over a filter membrane. The filter membrane has pores of a particular size. Particles larger than the pores will be unable to pass through the membrane, while particles smaller than the pores will pass through unhindered. Additionally, all liquids will pass through. The final result of a filtration process is a collection of residue on the filtration membrane. This residue is therefore effectively separated from the rest of the mixture that passed through the membrane.
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.
Enzymes play a surprisingly important role in modern industry, and are essential to the production of more commercial products than one would initially consider. Enzymes are proteins that speed up reactions and improve yield by increasing available precursors for downstream reactions. Perhaps the most obvious use for enzymes in industryisthe production of cheese, bread, and alcohol. In these traditional applications the enzymes are part of microbial machinery such as bacteria or yeast. Over time scientists have been able to isolate specific enzymes and to understand their catalytic functions well enough to incorporate them with or without their microbial hosts into a wide variety of somewhat surprising situations. For example, enzymes are used in the production of textiles, detergents, biofuels, and pharmaceutical products. Large quantities of desired enzymes are required for these applications, and they need to be available in the purest form possible. The purity of enzymes in industry is particular important for pharmaceutical applications where the products as well as the process are susceptible to review and control by regulatory associations. Batches of enzymes in industry undergo regular process validation to ensure batch-to-batch consistency.
The industrial centrifuge plays an integral role in the production of more things than one would initially expect. It is a commonly used tool in the food and agricultural sector, At pharmaceutical and biotechnology companies, for environmental management, and in the chemical industry. The word industry conjures up images of combination and creation—adding materials together to produce a final product. However, the separation of materials is just as important as the combination of materials. We can't create a new product until we have pure reactants to work with. This is especially important in the pharmaceutical and biotechnological realms, where reactant purity is essential to the production of a product that is safe for human consumption. This is where the centrifuge comes in. The centrifuge is used to separate heterogeneous mixtures into components varying by density.
An elution buffer plays an essential role in every immunoprecipitation protocol or assay that requires the release of a target antigen from a capture antibody. Elution buffers are necessary in protocols utilizing a stationary affinity column, and are also required in protocols using mobile solid supports in solution.
A cell lysis buffer is a critical first component to any isolation protocol. It is fundamental to the first step of protein or nucleic acid extraction as it aids in the chemical breakdown of cell membranes and compartments, enabling target molecules to escape. There are many types of lysis buffers; most are easy to make, but most are also commercially available. They are often included in kits for immunoprecipitation, co-ip protocol, nucleic acid isolation, and others. When using a lysis buffer for protein capture it is a good idea to add protease inhibitors prior to use in order to protect proteins.
The sandwich ELISA is a type of Enzyme-linked immunosorbent Assay that uses two antibodies: a capture antibody and a detection antibody. The purpose of any ELISA is to detect the presence of a target antigen in a sample. In a sandwich ELISA the target antigen is bound between a capture antibody and a detection antibody. The capture antibody is immobilized on a surface, while the detection antibody (conjugated to an enzyme or fluorophore label) is applied as a last step before quantitation.
The force of gravity will cause sedimentation of particles from a heterogeneous mixture; larger and denser particles sedimentfaster than the smaller and less dense particles. This phenomenon is useful for separating heterogeneous solutions into independent components, and for the isolation and enrichment of target molecules, cells, and cell organelles. Differential centrifugation accelerates the separation process by introducing centripetal forces many times greater than gravity. The precipitated particles form a pellet at the bottom of the tube during centrifugation. The rate of sedimentation is dependent on the size and density of the particles, so centrifugation can be used to isolate target particles simply by controlling centrifugal force or the rate of centrifugation. The rate of centrifugation is reported as angular velocity by revolutions per minute (rpm) or as acceleration(g). RPM is dependent on the radius of the rotor in the centrifuge.
Chemiluminescence and fluorescence seem like they are the same thing, especially when using them as tracking strategies for magnetic separation in biosensors or in-vitro diagnostic assays. But, they are not the same. Yes, they both give off a photon as an electron relaxes from a higher energy state to a lower energy state, but the difference lies in the method used to excite that electron to a higher energy state in the first place. In fluorescence the electron is kicked up to a higher energy state by the addition of a photon. In chemiluminescence the electron is in a high-energy state due to the creation of anunstable intermediate in a chemical reaction. Light is released when the intermediate breaks down into the final products of the reaction.