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Biotinylation Kit

What is biotin labeling?


Biotinylation 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.

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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The purpose of antibody dilution and how to do it

Antibodies are a key component to many biotechnical applications. They are most often used for immunoassays such as ELISA, cell and tissue staining, protein quantification such as western blot, and cutting edge sensor development. Verified antibodies are easily purchased from commercial vendors. These antibodies can be monoclonal or polyclonal, and can come as a lyophilized powder or as a premixed solution. All of these details must be considered when choosing which antibody to purchase because they all have an effect on the antibody concentration and dilution process. 

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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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Synthesis of fluorescent nanoparticles for bioimaging

Fluorescent nanoparticle is a general term that many people assume means any nanoscale material that produces fluorescence upon excitation with incident light. However, there is actually a thing called a conjugated polymer nanoparticle (CPN) that is very different from the fluorescent dyes (think Alexa fluorophores) that many of us are used to. These CPNs produce higher intensity fluorescence than dyes (up to 1000x brighter!), are stable and much less susceptible to quenching, and don’t contain toxic cadmium like quantum dots. These conjugated polymer fluorescent nanoparticles will likely be at the forefront of future bioimaging methods. 

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Protein Purification Methods

Protein Purification is essential to understanding the structure and function of various proteins. It is also important for the development of clinical biosensors, which rely on stocks of pure protein as detection agents. There are many methods of protein purification, which are discussed below along with how protein purification works, the steps involved, and how to increase yield in your protein purification protocol

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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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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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Cell centrifugation

It is useful, and often necessary, to break apart the cell and separate the cellular organelles into individual fractions for further study. Once the organelles are separated it is easier to identify pathways of disease or basic biochemical functions within the cell. One easy, and relatively gentle (if performed properly) method to do this is via cell centrifugation. This process involves three main steps: homogenization of the cellular extract, differential sedimentation, and density gradient centrifugation. The homogenization step breaks apart the cell membranes and releases the organelles into one big cellular soup. Then, the first step of centrifugation begins with differential sedimentation. This results in a rough separation of organelles. The fractions are further separated into clean fractions by density gradient centrifugation, which uses a material gradient (often sucrose) to help separate the organelles by density during centrifugation. 

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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 fusion protein and glutathione is what makes it useful for affinity chromatography. Once a capture GST fusion 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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Enzyme Immunoassay

An enzyme immunoassay is used to detect the presence of a target protein in a biological sample. A more common name for an enzyme immunoassay is the enzyme linked immunosorbent assay (ELISA). These assays are the cornerstone of most established clinical tests, and are typically the gold star diagnostic method. The ELISA is also used in many research environments because of its reliability and sensitivity. It is commonly able to detect protein down to concentrations as low as single picograms per milliliter. The sensitivity depends on the antibody-antigen pair and on the performance of the enzyme-substrate pair. Enzyme immunoassays are labeled assays because they don’t detect a protein binding event (between antibody and antigen) directly. Instead, these assays rely on an enzymatic reaction between an enzyme-conjugated label and a substrate to produce a colorimetric, chemiluminiscent, or fluorescent product. These products are then detected via a spectrophotometer or fluorometer and compared to a standard curve to produce quantitative concentration information about the target protein in the experimental sample.  

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