Purification and identification of protein of interest, SDS –PAGE of the purified recombinant protein

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Chromatography to purify proteins of interest depends on a protein's chemical or physical properties, such as molecular weight, electrical charge, or solubility. Green Fluorescent Protein (GFP) is extremely hydrophobic compared to bacterial proteins. This unique characteristic of GFP enables the purification of GFP from bacterial cell proteins using hydrophobic interaction chromatography (HIC). When placed in a buffer containing a high concentration of salt, the HIC matrix selectively binds hydrophobic GFP molecules while allowing the bacterial proteins to pass right through the column. Then, simply lowering the salt concentration of the buffer causes GFP to elute from the column in pure form. A colony of transformed bacteria are placed in liquid cultures overnight then lysed to release their cellular contents. GFP is purified from the bacterial contaminants using the HIC columns provided in the kit. Electrophoresis is the process of moving charged molecules in solution by applying an electric field across the matrix (gels made of polyacrylamide or agarose). Electrophoresis is used for analysis and purification of proteins and nuclei acids. Polyacrylamide is the most common matrix for separating proteins. In sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis separation, the migration is determined by molecular weights. SDS is an anionic detergent that denatures proteins by wrapping the hydrophobic tail around the polypeptide backbone. SDS also disrupts hydrogen bonds, blocks hydrophobic interactions and substantially unfolds the protein molecules. The proteins are totally unfolded when a reducing agent such as dithiothreitol (DTT) is added. DTT cleaves any disulfide bond between cysteine residues. The SDS-denatured and reduced polypeptides are separated by molecular weight.

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