Techniques for Separation of macromolecules in Bio Lab

Since there are thousands of different macromolecules in each cell, purification of a specific one from all the others requires powerful separation techniques, such as chromatography and electrophoresis. Both of these approaches take advantage of physical and chemical properties that differ between the individual macromolecules.

1) In gel electrophoresis, the macromolecules are placed in a solid matrix, called a gel, which is under a liquid buffer. An electric field is applied to this system, and since biological macromolecules carry ionic charges, they will be attracted towards one pole of the electric field and repelled by the opposite. Thus, macromolecules characteristically migrate in either direction in the field. The migration speed is determined by the charge-to-mass ratio of the macromolecule.

1A) In a flat gel, also called a horizontal or submarine gel, electrophoresis system, an agarose gel lies horizontally below the electrophoresis buffer. This technique is mainly used to separate large nucleic acids (DNA and RNA).

1B) A vertical electrophoresis system holds a polyacrylamide gel in the vertical position, and is mainly used to separate proteins or small-sized nucleic acids.

2) Chromatography is a family of methods used to separate macromolecules through their relative affinity to a stationary phase (generally, solid chromatography beads) and a mobile phase (generally, an aqueous buffer). The chromatography beads are loaded into a tube, called a chromatography column, and buffer is dripped, or pumped, through the column to carry the macromolecules along. The macromolecules with the least affinity to the chromatography beads travel through the column the quickest, while the macromolecules with the most affinity to the chromatography beads are the last to leave the column. Some chromatography beads separate by charge (ion exchange chromatography), by hydrophobicity (hydrophobic interaction chromatography), or by a specific property of that protein (affinity chromatography). Macromolecules can also be separated by size otherwise known as size exclusion or gel filtration chromatography. Generally, macromolecules separate from each other more cleanly when the chromatography beads are very small, but smaller size creates a backpressure and slows the rate at which the mobile phase can drip through the column. To overcome this limitation, high performance (or high pressure) chromatography (HPLC) uses high-pressure pumps and metal-jacketed columns to operate at high pressures and speed up the process.

3) A fraction collector collects the released mobile phase (eluent) of a chromatography column. It automatically measures a programmed volume (sometimes by the number of drops of liquid) into a line of test tubes or microcentrifuge tubes.

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