Polyacrylamide Gel Electrophoresis (PAGE)

Definition and applications-1 Electrophoresis is the technique of separating molecules with a certain charge from an electric field by the help of the electric field. Polyacrylamide Gel electrophoresis is the most commonly used type of electrophoresis. SDS-polyacrylamide gel electrophoresis is a method in which the charged molecules are loaded to a gel and subjected to an electric current to separate molecules according to their molecular weights. SDS- polyacrylamide gel electrophoresis is widely used in blochemistry.

Definition and applications-2 The polyacrylamide gel contains acrylamide, bisacrylamide, a denaturing agent such as SDS, and a buffer with known pH. Polyacrylamide gel electrophoresis uses two gels called stacking gel and resolving gel. The polymerization of the gels is provided by agents such as ammonium persulphate and TEMED. After the stacking gel is poured into the glasses, butanol can be added to prevent bubble formation or flatten the surface. Although the ratio of bisacrylamide to acrylamide may vary according to the molecule to be analyzed, the concentration of acrylamide is usually 1 part of bisacrylamide to 35 parts of acrylamide. Acrylamide concentration in the gel may vary and different concentrations are used for different molecular weight proteins.

If the gel is prepared manually, it is poured between the glasses in the gel pouring system using special glasses with specific spacers. Firstly, the resolving gel is poured into the gel pouring system and then the stacking gel is poured and a comb i attached to the stacking gel. The thickness of this comb must match the thickness of the spacer on the glass and this determines the thickness of the gel.

Preparation of gels: Concentrations of agents used in polyacrylamide gel electrophoresis: For example, below are the concentrations of agents necessary for the separating gel containing 9.5% of acrylamide. By varying the acrylamide concentration of the resolving gel, gels having different pore sizes can be obtained according to the molecular weight of the molecule to be analyzed.

Resolving gel: 5.55 ml H2O 3.3 ml 1.5 M Tris-HCl pH 8.8 4.2 ml Acrylamide 132.5 ul %10 SDS 132 ul %10 APS 6.5 ul TEMED Stacking gel: 4.25 ml H2O 1.8 ml 0.5 M Tris-HCl pH 6.8 0.95 ml Acrylamide 70.5 ul %10 SDS 35.5 ul %10 APS 3.5 ul TEMED Polyacrylamide gels can be prepared manually in the laboratory as well as commercially available. One has to consider the molecular weight and properties of the biomolecule of interest, to order desired percentage of pre-made ready gels.

Preparation of samples loaded to the gel: Samples containing equal amounts of protein are mixed with loading buffer and denatured in 100 ° C water bath for 4 minutes. The loading buffer usually contains β-mercaptoethanol (β-ME, 5% volume), dithiothreitol (DTT, 10 mM) or dithioerythritol (DTE, 10 mM) in buffer.

The glasses are placed in the electrophoresis tank and electrophoresis buffer is placed in the tank. Samples are loaded with automatic pipette. After the loading of the samples is finished, the lid of the tank is closed and electrophoresis process is started at 100 V. The electrophoresis is completed in about 2-3 hours depending on the size of the gel.

Coomasie Blue staining: With the prepared coomasie brillant blue G250 dye, the gel is waited for about half an hour at 20-25 ° C and the gel is washed with distilled water after the staining process is completed.