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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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Immunoaffinity chromatography

Immunoaffinity chromatography is a method for separating target antibodies or antigens from a heterogenous solution. It is column-based, which means that the solution is flowed through a column and eluted at the other end. The column is pre-functionalized with the capture antibody or antigen. The target protein is adsorbed onto the resin-bound capture protein and is retained in the column while the remaining solution is eluted. The fraction containing the target protein is later eluted and purified.

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Purified Proteins for drug discovery

Modern drug discovery utilizes libraries of purified proteins. These proteins are screened by libraries of small molecule drug precursors. This combinatorial screening process greatly speeds up the identification of new drug molecules.

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Size exclusion chromatography columns

Size exclusion chromatography is used to separate molecules by size, molecular weight, and hydrodynamic volume. The technique can be used with proteins, polymers, and other macromolecules. It can also be used for buffer exchange or desalting a sample. The principle behind size exclusion chromatography is simple, but the technique only works when the correct resin-bound column is matched to the experimental goal.  

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Protein Extraction

Proteins are essential components of cells, tissue, and organisms. These macromolecules are made of long strings of amino acids arranged specifically into three dimensional configurations. The side chains of these 22 amino acids create pockets of potential for chemical interactions as the polypeptides fold into their tertiary structures and interact with each other. Proteins initiate and mediate the thousands of biochemical pathways that govern an organism’s function. The careful study of proteins can reveal information about the function of our bodies, the pathways of disease, and the expression of the genetic code.

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