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Protein Purification Buffer

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.

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Cell sorting techniques

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.

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Silica magnetic beads

Chemistry of the silica magnetic beads

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Direct Elisa and Indirect Elisa

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.

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Magnetic beads immunoprecipitation

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

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Magnetic Cell Separator

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.

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Magnetic Microbead

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.

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Magnetic beads DNA

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. 

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Enzymes in Industry

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.

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The industrial centrifuge

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.

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