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Protein Extraction Buffer

Proteins are fundamental building blocks for life. All tissues and organisms are made up of protein, and all of the work performed inside and outside of cells is mediated by protein signaling cascades. Proteins are polymers of amino acids with three or four layers of organized structure. Primary structure is defined as the linear order of amino acids. This is dictated by the genome: the code is transcribed from DNA and translated into the string of amino acids. Secondary structure is thought of as two basic forms: a beta sheet or alpha helix. The string of amino acids adopts the conformation that allows the lowest energy state. Beyond the sheets and helices, the chain can take other twists and turns to fold into a shape known as its tertiary structure. Some proteins are actually made up of two or more subunits of individually folded amino acids strands. The complexing of protein subunits to form one functional protein is called quaternary structure. All of this folding is extremely important to the character and function of each individual protein because it results in certain side chains of amino acids being located on the exterior or interior of the protein. Importantly, the folds create binding pockets where key amino acids are located to create a unique chemical landscape that allows the protein to bind to other proteins and carry out its job in a signaling cascade.

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Detergent Compatible (DC) protein assay

A protein assay is used to quantify the amount of total protein in a sample. There are two main ways to do this, and both involve a color change as an indicator of protein presence, which means they are colorimetric assays:

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Bacterial Antigens

An antigen is defined as anything that causes an immune response in another organism. This immune response can be a simple increase of inflammatory factors, or it can be an activation of the adaptive immune system and creation of antibodies. Antibodies have two or more specific paratopes, or antigen recognition sites, that identify and combat the invading antigen. The number of antigen recognition sites is dependent on the antibody class. The word “antigen” can also refer to any protein of interest detected by a bioassay or biodetection platform. In the case of a bacterial antigen, we are referring to surface proteins, lipopolysaccharides, and peptidoglycans on the bacterial cell wall; these structures help bacteria invade other organisms by gaining access between epithelial cells. While surface structures help bacteria infect other organisms, they are also a detriment to the bacteria because they  also serve as a unique tag that antibodies and bacteriophages can recognize. Bacteriophages are viruses that attack bacteria. Both antibodies and phages are being used by scientists to develop new biodetection and biosensing platforms for rapid detection of bacterial antigens in the environment and in clinical samples.   

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Protein purification services

Protein purification services are available for anyone who is in need of a custom antibody or recombinant protein for research and development purposes. If your laboratory is not equipped to produce recombinant proteins in house, then this may be an attractive option. These services require you to provide a sequence and preferred expression system; in turn they will deliver a quality controlled protein with accompanying documentation to you within just a few weeks.

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ELISA Troubleshooting

The ELISA or Enzyme-Linked Immunosorbent Assay is the most established method of protein detection and quantification. The technique is commonly used in a wide variety of applications spanning from the basic research bench to clinical laboratories. The ELISA is a labeled assay, which means that it requires the use of a label to detect an antibody-antigen binding event. The label is commonly a fluorescent probe, chemiluminescent system, or enzymatic colorimetric reaction.

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Purification Techniques

The earliest chemists were on the hunt for new elements to add to the periodic table. Most of the chemistry that they were interested in doing was purification with the end goal of reaching a pure elemental substance. These chemists relied on a litany of methods—filtration, evaporation, distillation, and crystallization were some of the most used purification techniques for these discoveries. As the chemists were defining the elements, the biologists were trying to understand the human body, the cell, cellular organelles, and microbes. The point here is that in order to develop anything new we must first understand what everything is made of at the most basic and pure level. In modern science this means that we are trying to define matter beyond subatomic particles and we are attempting to map out every molecular pathway of disease. Our efforts to define complex systems by their purest constituents are rewarded by deep understanding and an ability to mimic, to engineer, develop, and create.

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Protein Purification with a protein A column or bead

If you look closely at the product information for many commercial antibodies, you will see that they are protein A purified. Protein A is a surface protein that was originally found in the cell wall of staphyloccoccus aureus bacteria. On the surface of bacteria it serves as a defense against the host immune system and allows the bacteria to survive longer and be more virulent. Protein A binds the Fc portion of IgG antibodies.

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Single Domain Antibody

Until this point we have been thinking about antibodies as one of the five classes, IgG, IgE, IgD, IgA, or IgM. The basic unit of each antibody class is a Y structure, where the base of the Y is known as the Fc region and the arms are the Fab region. The entire IgG antibody is composed of four polypeptide chains (two heavy and two light). The Fc region is composed only of heavy chain, and the variable Fab region is built with heavy chain and light chain. The Fab region is where all of the antigen-binding  occurs because the paratope, or antigen recognition site is located at the tip of each of the two arms of the Y. A single domain antibody paratope is made solely of a single heavy chain.

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The step-by-step process of protein purification

Protein purification is the processes of isolating a protein of interest from its environment. In other words, from the other natural molecules surrounding the proteins in the natural niche in a host organism, or from a cell culture grown in a laboratory. Our protein purification handbook  explains that there are several available techniques and many options to consider, but the general procedure is the same. 

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Protein expression and purification

Proteins are constantly being created by the cellular machinery of living organisms. This article will first summarize the process as it occurs in a natural organism, and then discuss how protein expression and purification occurs in a laboratory setting for the generation of recombinant proteins.

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