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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 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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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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Cell based assays

Cell based assays are used to quantify cellular function, measure how stimuli affect cells, or to localize an effect within the cell. The cells are live and intact, and require the use of fluorescent tags and chemiluminescent or colorimetric enzymes. The quantification is performed by flow cytometry or microscopy. This is very different from studies of protein or nucleic acid which require destruction of the cell and isolation of those components from cell lysate. A cell based assay is conducted entirely within live, intact cells. The goal is to understand a cellular process, localization of a molecule or drug to a cellular compartment, or to measure how cells react to a substance. Cell based assays are usually performed in tightly controlled cell lines to test for a wide range of behaviors:

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Competitive ELISA

The enzyme linked immunosorbent assay (ELISA) is a gold standard method for protein and antibody detection. It is routinely used for clinical lab work and is often used in research and development. The assay is based on the lock-and-key specificity between an antibody-antigen pair. Antibodies are naturally made by the adaptive immune system to recognize a specific piece of an antigenic protein. This is what allows the body to identify and fight invading pathogens so effectively. Since antibodies have such a fundamental role in most biosensing and diagnostic systems it is very easy to purchase them or have them custom-made to recognize a protein of interest. The ELISA assay is performed in 96- or 384-well plates that are treated to favorably adsorb antigen or antibody depending on the type of ELISA being performed. The ELISA assay is built up from the bottom of each well; it doesn’t float freely in the well. As a result, the unbound proteins are easily washed away and only the target remains to be quantified.

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