In this animation produced by WGBH and Digizyme, Inc., learn how protein purification separates and isolates a protein of interest from other contents of transformed bacterial cells that have undergone lysis. The animation explains the steps involved in a technique called hydrophobic interaction chromatography (HIC), which ultimately yields a solution that is highly enriched with the protein of interest.
Protein purification is a series of processes designed to isolate, or purify, a single type of protein from a complex mixture of proteins and/or other cell parts. Purification is done for analytical or preparative purposes. Analytical purifications produce a relatively small amount of a protein for a variety of research purposes, including identification and study of the protein’s structure. Preparative purifications produce a relatively large quantity of purified proteins that can later be used for particular purposes, such as in industrial enzymes or medicines.
Chromatography refers to a variety of techniques designed to separate the components of a mixture. In chromatography, a mobile phase, typically a gas or a liquid containing the molecules to be separated, is passed over a stationary phase. This stationary phase acts as the separating medium, or resin as it's commonly called. In column chromatography, a matrix is used as the resin. The interaction of the molecules with resin affects the rate of movement of molecules in the mixture.
This lab animation shows how a hydrophobic stationary phase is used to purify a hydrophobic red fluorescent protein (mFP) molecule—the protein of interest. This variation of column chromatography is called hydrophobic interaction chromatography (HIC). Hydrophobic molecules naturally repel water, while hydrophilic molecules are attracted to water. Proteins and other large molecules often have some regions along their structure that are hydrophobic and others that are hydrophilic. If these proteins are placed in water, the molecules distort, or change conformation. The hydrophobic regions tend to hide from water, turning inward, while the hydrophilic regions move outward. HIC uses as the resin a bed of small hydrophobic beads that are tightly packed inside a plastic or glass cylinder. Because the red fluorescent protein molecules are highly hydrophobic in a high-salt medium, they stick to the resin. This allows all the hydrophilic molecules to be washed away as they continue down the column without sticking to the resin.
Once the mFP is trapped in the resin bed, the column can be washed of moderately hydrophobic molecules using a lower-salt concentration buffer. Finally, the mFP is washed off the column using a buffer with very low salt. Under low-salt concentration, the protein starts slowly folding back to its original conformation. The hydrophobic regions of the molecule move toward the interior of the molecule, allowing the movement of the mFP into the buffer and off the column. The resulting solution, or eluate, contains the protein of interest and may contain other molecules with similar hydrophobicity.
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