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Protein Isolation protocol

A protein isolation protocol aims to safely and efficiently separate a protein that you are interested in from a mixture. Isolation of proteins is done from mammalian, insect, plant, yeast, or bacteria samples. 

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Protein A vs Protein G

Protein A vs Protein G, what is the difference? They are both bacterial cell wall proteins that have primary binding sites for mammalian immunoglobulin G (IgG) antibodies, including human IgG. Protein G was first isolated from Streptococcal bacteria strains C and G. Similarly, protein A was originally found on the cell wall of the bacteria Staphylococcus aureus.  These proteins have primary binding domains for the Fc region of (IgG) antibodies, but can also recognize the Fab region of certain IgG subclasses described in more detail below.. For the bacteria this is useful because binding IgG’s at the Fc region prevents macrophages from recognizing them, which in turn prevents phagocytosis of the invading bacteria by the host immune system. For scientists this IgG binding can be used for probing in experiments such as purifications or immunoprecipitations.

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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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Gst tag sequence for protein isolation

Glutothione S-transferase is a 26 kDa protein that is used as an affinity tag for protein isolation in pull-down assays. The GST tag has specific affinity for the protein glutathione. This means that glutathione can be attached to columns or magnetic beads and used to isolate any protein that has been modified with the gst tag sequence. The modification of proteins with the gst tag sequence is performed in host organisms and results in fusion proteins that consist of the target protein joined by a linker to the 220 amino acids that compose the gst tag. 

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BCA protocol for protein quantification

The BCA assay is used to quantify protein concentration by using bicinchoninic acid to identify copper ions reduced by protein in a biuret reaction. The BCA protocol requires a working solution mixed with the sample; when protein is present, the reaction produces a purple color that absorbs light at 562 nm and is quantified with a spectrophotometer. The BCA assay is similar to other protein quantification assays such as Lowry or Bradford assays. However, the biuret reaction of the BCA assay occurs between the nitrogens on the peptide backbone and copper as well as nitrogens on the amino acid side chains. The fact that the peptide backbone participates in the reaction means that the BCA assay is more consistent between proteins and is less dependent upon amino acid composition. The BCA protocol is simple and quick. If the sample is heated to 37°C, then the incubation time is only 30 minutes, and the absorbance measurement takes only a few minutes. The BCA assay is an excellent method for quantifying total protein concentration after biomagnetic protein purification.

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Protein Extraction Protocol

Proteomics is the study of the protein in an organism. Protein is a fundamental building block of life, and proteins are the workhorses within and between cells. Biochemical pathways are built out of enzymes and ligands—without them nothing would be accomplished; plants wouldn’t produce glucose, animals wouldn’t be able to digest food, the immune system would cease to exist, and all other biological processes would grind to a halt. The fundamental importance of proteins for life makes them an important topic of study. The first step in understanding protein structure and function is to extract them. Protein extraction is the process of isolating and purifying protein from samples of whole tissue, cell cultures, or biological fluids. The protein extraction protocol used is tailored to match the starting material and the end goals of the assay. Considering the goal of the experiment is extremely important when developing a protein extraction protocol because certain buffer choices (such as high salt, high detergent formulations) can ruin an experiment when higher order protein structure and function needs to be preserved.

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Sonication cell lysis protocol

Cell lysis is the first step of breaking the cell membrane that enables further study of the proteins, nucleic acids, and other molecules inside of cells. When cell lysis is successful, the undamaged contents of the cell escape through the damaged cell membrane. These contents are then separated out of the mixed sample and used for further study. The methods used for separation of the lysed cell contents are dependent on the goal of the study. Careful investigation of these inner workings can reveal disease patterns, improve our understanding of normal cellular function, and elucidate biochemical pathways and therapeutic targets. Protein isolation is different from nucleic acid separation, and the reagents used vary drastically. There are a few ways to lyse the cell membrane; these include mechanical disruption, liquid homogenization, freeze/thaw cycles, manual griding, and the use of detergents. Sonication cell lysis is an example of mechanical disruption used for releasing the contents of cells.  

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Types of antigen

An antigen is a substance that is capable of stimulating an immune response and activating white blood cells to produce antibodies. Antigens can be proteins or sugars that are located on the outer surfaces of cells. All cells have antigens including the ones inside the body, bacteria, and even viruses. The antibodies produced by the immune system are custom-fitted to the antigen that initially stimulated the immune response. The antibodies have an antigen recognition site (paratope) that has highly specific affinity for a region on the antigen called the epitope.  

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Antibody validation

Antibodies are naturally produced by the adaptive immune system in response to invading pathogens. The antibodies are made by immune cells to specifically recognize protein markers called antigens located on the outer wall or membrane of the pathogenic organism. It is this exquisite antigenic specificity that makes the adaptive immune system so remarkable in its ability to fight off a wide variety of diseases. It is also this specificity that makes the antibody-antigen interaction an attractive tool for the development of biological assays for the detection of active infection and disease.  

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BCA protein assay

The BCA protein assay is used to quantify total protein in a biological sample. BCA stands for Bicinchoninic acid, which is the key reagent used to produce a colored product. The purple colored product is analyzed in reference to a standard curve in order to quantify protein concentration. It is important to measure protein concentration after performing a protein extraction or purification, and prior to any type of labeling procedure. The protein concentration after extraction or purification may provide information about a biochemical pathway or a disease state. All commercially available proteins are accompanied by a product information sheet that has the results of a protein quantification method. This is particular important in antibody validation. It is important to know the protein concentration prior to any labeling step so you can ensure that the stoichiometric ratio between label and protein is optimal for clean and efficient labeling. It is equally important to know how much protein you are working with when designing biosensors so that you can define limits of detection and instrument sensitivity. 

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