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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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Elution Buffer

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.

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Cell Lysis Buffer

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.

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Isolation of CTCs from centrifuged blood and immunomagnetic separation

Circulating Tumor Cells (CTCs) are cancerous cells that dissociate from a tumor and circulate throughout the bloodstream. Therefore, the detection of CTCs in the bloodstream is an indicator of cancer progression and an early sign of metastasis. CTCs are not hematopoetic in origin, and they do not express the cell surface marker CD45. However, they do express the surface antigen EpCAM, which is commonly expressed on epithelial cells. Immunomagnetic separation methods take advantage of these surface markers to isolate CTCs from centrifuged blood samples.

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Process Validation Protocol

What is process validation and why do we do process validation?

Good manufacturing practice is an essential part of the production of human drugs, veterinary drugs, biological and biotechnology products, and pharmaceutical ingredients. These commercial processes are subject to regulatory oversight and must ensure that every aspect of the production process is carefully scrutinized. The purpose of process validation is to collect data and scientifically analyze the production process from conception to large scale production. An updated process validation protocol is essential to ensuring product quality and consistency. Many laws have been established to mandate process validation in order to protect consumers, especially in the case of pharmaceutical products.

Process validation in the pharmaceutical industry takes the same form as process validation in all other industries, but the stakes are higher because the product is made for human consumption. Moreover, pharmaceuticals are made to alter the natural biochemical pathways in the human body, so these chemicals must be formulated correctly and consistently every time. Additionally, the products must be stored properly and shipped under climate-controlled conditions in order to ensure efficacy once reaching the consumer.

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Biotinylation Kit

Biotinylation kit means attaching a biotin tag to a molecule. Biotin is a natural molecule that is also known as vitamin B7. It is an important component in a healthy diet, but it is also very useful in the laboratory as a method for protein conjugation. In the laboratory, he purpose of biotinylation is to create a controlled site for biotin-streptavidin affinity binding.

What is biotin labeling and how does streptavidin bind biotin?

The biotin fits exquisitely into a biotin-binding pocket in each of the four binding sites per streptavidin molecule, and it is held in place with hydrogen bonds. Additionally, once the biotin is bound, a conformational change in the streptavidin allows a small “cap” to close over the biotin in the binding pocket. As a result, biotin and streptavidin have an extraordinary affinity for each other (Kd=10^-15). With such a low dissociation constant, once the biotin and streptavidin are bound it is unlikely that they will dissociate. This affinity is resistant to changes in temperature, pH, and salt concentration and is extremely specific. It is often thought of as a nearly covalent bond. These properties make biotinylation a useful tool for engineers who are developing new purification and detection methods. A commercially available biotinylation kit makes the process even easier.

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Co-ip protocol: Co-immunoprecipitation

 

The difference between IP and coIP

Co-immunoprecipitation (coIP) is a protein extraction technique that specifically targets protein-protein interactions. It is slightly different from immunoprecipitation. Immunoprecipitation utilizes antibodies immobilized on a mobile support to capture target proteins. Co IP protocol takes this concept one step further by using antibodies to target not only the direct antigen that binds to the antibody, but also any protein that binds to the antigen and is pulled out with it. This makes co-ip protocol an ideal technique for studying protein complexes. The main concern when developing a co-ip protocol is to ensure that the lysis, wash, and elution buffers do not denature the proteins. Otherwise the tertiary structure of the proteins will deteriorate and the protein-protein interaction may be altered or completely lost.

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Antibody Purification

Antibodies are an important part of the immune system. When the body is infected with an antigen, the immune system generates an antibody specific to that antigen. The techniques that are routinely used in biotechnology capitalizes upon this natural immune process. Antibodies are used in many research applications as well as in immunoassays for disease detection. We use the specificity of the antigen/antibody binding for immunoprecipitation and ELISA assays. We use flurophore-conjugated or enzyme-tagged antibodies for labeling molecular targets on individual cells and whole tissue. We use antibody purification to obtain antibodies for biosensors to detect disease. These antibodies, depending on the application, are commonly obtained by antibody purification from humans, rats, rabbits, mice, and chicken. 

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GST fusion protein

A gst fusion protein (Glutathione-S-transferase) is useful for affinity chromatography and immunoprecipitation. The natural form of GST is an enzyme that catalyzes the protective mechanisms of glutathione. Glutathione is an antioxidant that prevents cell damage by reactive oxygen species. However, the GST fusion protein is not natural. It is a genetically engineered protein that has become a useful biotechnological tool.

The affinity between the GST protein and glutathione is what makes it useful for affinity chromatography. Once a capture GST protein is created, it can be used to capture a target protein, and the whole complex can be isolated by running it through a glutathione column. The complex is then eluted by adding an excess of glutathione, which out-competes the bound glutathione and fills the GST binding site resulting in release from the column. 

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