Purpose of Protein Expression Analysis
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Protein Expression
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The expression of proteins is very simply the production of a protein from a defined amino acid sequence, which is in turn determined by a defined gene sequence. Understanding these sequences allows an investigator to modify a protein in various way--for example, by placing a fluorescent tag on the protein so it can be detected, or by removing portions of the protein that may interfere with its production or processing by a cell.
Methods and Tools for Analysis of Protein Expression
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Protein expression is analyzed by using highly sophisticated and specialized technologies and procedures, such as ELISA, Immunoassays, mass spectrometry, and imaging. In the majority of cases the protein is indirectly detected--for example, by using an antibody that binds to it and then using a secondary detection system to visualize it. A basic protein detection technique is that of Western blotting, during which a protein is run through an agarose gel and then transferred onto a membrane. It is then "probed" by allowing an antibody to bind to it, and the antibody is then reacted with a chemical solution, such as a silver stain, to produce an image of the protein's location on the blot. This gives an indication of the size of the protein and whether it is present as multiple or single units. Another commonly used method is fluorescent detection. The binding of an antibody tagged with a fluorescent particle to the protein will allow it to be visualised by microscopy.
The Purpose of Analyzing Protein Expression
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Studying the expression of a protein gives an investigator an indication of where it is being produced (i.e., its expression pattern and localization) or its quantities (i.e., its tissue or cellular levels). This information is used in diagnostic pathology in, for example, the detection of urinary protein to determine whether a patient has kidney diseases such as renal tubular disease or diabetic nephropathy. In genetic laboratories gene cloning experiments usually generate recombinant (genetically-altered) proteins. The analysis of these synthetic molecules is crucial in verifying gene sequences or checking for mutations that may or may not be desirable. For example, vaccines are proteins produced by artificially-modified units of genes. These proteins must be analyzed carefully to ensure that the genes were altered in the correct and desired way, so as to avoid unwanted side or toxic effects.
Protein Expression Analysis by Western Blotting
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The analysis of protein expression using western blotting is a very routinely performed laboratory procedure in order for scientists to evaluate gene cloning and recombinant protein production. For example, in molecular biology and genetic engineering, genes from different species (such as mouse and human) are joined together and placed into a bacterial cell, which is capable of expressing the protein. In order to ensure that the correct protein is being generated, its expression must be analyzed by western blotting. Western blotting is also used in medical pathology to diagnose mad-cow disease and HIV.
Protein Expression Analysis by Flow Cytometry
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Flow cytometry requires cells to be stained with a fluorescent antibody, which binds to a protein expressed on the surface of, or inside, a cell. It is used in pharmaceutical research to find molecules (cytokines) that are unique to a specific set of cells and involved in cell signaling and communication. This is important so that the effect of drugs on normal cell physiology and function can be monitored during drug development and clinical trials.
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