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Protein Detection

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Purifying, Detecting and Characterizing Proteins

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Purifying, Detecting and Characterizing Proteins


Isolation of proteins is based on their physical and chemical properties . The most common techniques for purifying and analyzing proteins are centrifugation, electrophoreisis and chromatography.
Centrifugation: With this technique proteins are separated based on their rate of movement in an applied electric field.
SDS-Polyacrylamide Gel Electrophoresis:  With this technique polypeptide chains are resolved differing in molecular weight by 10 percent or less.
Liquid Chromatography: This technique seperates proteins based on their rate of movement through a column packed with spherical beads.  Proteins that have different masses are resolved on gel filtration columns and proteins that have different ligand binding properties are resolved on affinity columns.
Many different assays are used to detect, identify and quantify proteins.  Assays which are most sensitive use a light-producing reaction or radioactivity to generate a signal. Other assays produce an amplified colored signal with enzymes and chromogenic substrates.
Antibodies are used as powerful reagents to detect, quantify and isolate proteins. They are used in affinity chromatography and combined with gel electrophoresis in Western blotting.
Autoradiography detects radioactive labeled molecules in cells, tissues or electrophoretic gels.
X-ray crystallography, NMR spectroscopy and cryoelectron microscopy are all techniques that obtain the 3D structure of proteins. X-ray crystallography provides the most detailed structures, but requires protein crystallization.  Cryoelectron microscopy is particularly useful for large protein complexes which are difficult to crystallize. Only relative small proteins are amenable to NMR analysis.

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