Gel Electrophoresis What is Gel Electrophoresis A method

Gel Electrophoresis

What is Gel Electrophoresis �A method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.

What is Gel Electrophoresis �Uses of gel electrophoresis: Separation of proteins by charge and/or size Separation of a mixed population of DNA and/or RNA fragments by length

Principles of Gel Electrophoresis �An electric field is used to drive the movement of molecules (such as DNA) through a gel made of agar or polyacrylamide. �The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source.

Principles of Gel Electrophoresis �When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel

Gel Electrophoresis of DNA The phosphate (PO 4 -) groups of the nucleotides that make up the fragments of DNA & RNA give them a negative charge. Thus, when an electric current is applied, the DNA/RNA fragments migrate towards the positive pole

Gel Electrophoresis of DNA

Protein Electrophoresis �Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the polyacrylamide gel at similar rates, or at all, when subjected to an electric current �Proteins therefore, are usually denatured in a detergent such as sodium dodecyl sulfate (SDS) that coats the proteins with a negative charge.

Protein Electrophoresis �The amount of SDS bound to the denatured protein is relative to the size of the protein (usually 1. 4 g SDS per gram of protein) �The resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio.

Protein Electrophoresis Denaturation changes the protein’s shape from complex to long and rodlike, thus, the rate at which the SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape

Visualization & Analysis �After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. �DNA may be visualized using ethidium bromide which causes the DNA fragments to fluoresce under ultraviolet light

Visualization & Analysis �Protein may be visualized using silver stain or Coomassie Brilliant Blue dye. �Other methods may also be used to visualize the separation of the mixture's components on the gel. If the molecules contain radioactivity an autoradiogram can be recorded of the gel.

Analysis �DNA bands can be cut of a gel (post electrophoresis) and can then be dissolved to retrieve the purified DNA.

Web Links! Gel Electrophoresis Virtual Lab Protein Electrophoresis Video DNA Gel Electrophoresis