Polyacrylamide gel electrophoresis
In biochemistry, polyacrylamide gel electrophoresis ( PAGE ) is a method for separating molecules, especially proteins and nucleic acids .
Production of collecting and separating gel
For electrophoresis , the gel is produced by radical polymerization from the substances acrylamide and N , N '-methylene bisacrylamide (usually in a ratio of 37.5: 1). The latter is used to cross-link the otherwise linear polyacrylamide chains. This is the only way to create a rigid gel. The average pore diameter of a polyacrylamide gel is determined by two parameters, the total concentration of acrylamides (% T with T = total concentration of acrylamide and bisacrylamide) and the concentration of the crosslinker bisacrylamide (% C with C = bisacrylamide concentration). The pore size is inversely proportional to% T. The pore size decreases with increasing% T. At% C, a concentration of 5% results in the smallest pores. The influence of the bisacrylamide on the pore size results in a parabolic curve with the vertex at 5%.
If N , N '-diallyl tartardiamide is used instead of N , N ' -methylene bisacrylamide , then the gel can be dissolved again by glycol cleavage after the separation run with sodium periodate solution , which facilitates the investigation of the separated substances.
The gel consists of a separating gel over which a low layer of collecting gel is pipetted. The mixtures for both are first prepared separately depending on the desired composition. The pH of the buffer used, the concentration of acrylamide and the bisacrylamide content in the starting mixture determine the separation properties of the gel. In batch electrophoresis , different buffers are used to make a stacking gel and a separating gel.
The separation gel mixture is mixed with the radical starter ammonium peroxodisulfate (APS) and the polymerization catalyst tetramethylethylenediamine (TEMED) and quickly poured between two sealed glass plates that are separated from each other by a spacer (the distance is about 1.5 mm). No bubbles should remain in the gel. The panes must be absolutely clean and free of grease.
If you use riboflavin instead of ammonium peroxodisulfate , you can determine the point in time of the polymerization yourself with the help of bright, blue-violet light, which allows a greater amount of time for pouring the gel.
The toxicity of the unpolymerized acrylamide also requires the use of gloves. However, once the gel has fully polymerized, it is largely harmless to health. The separating gel is then covered with a little water, isopropanol or butanol , on the one hand to smooth the gel boundary and on the other hand to exclude contact with oxygen, since the oxygen in the air would intercept the radicals necessary for polymerisation and thus interfere with correct polymerisation of the gel.
The time it takes for the gel to polymerize depends on the concentration of the free radical initiator and the polymerization catalyst. As soon as the polymerization is complete, the water or alcohol can be poured off or suctioned off. APS and TEMED are then added to the stacking gel and pipetted over the separating gel. A special comb is inserted to obtain pockets for filling in the samples. A layer of water / alcohol is superfluous thanks to the inserted comb. After curing, the comb can be removed.
Procedure
The finished polyacrylamide gel is clamped in an electrophoresis apparatus.
The samples are pipetted into the recessed pockets. A bag is usually associated with a reference, e.g. B. a protein standard containing several proteins of known size filled. A visible, negatively charged dye (e.g. bromophenol blue ) is also added.
Since the molecules to be separated have a charge, they are "pulled" through the polymer when a voltage is applied (10 to 20 V / cm gel length at 1 mm thickness) and are separated electrophoretically depending on the charge, size, molar mass and nature of the separation medium (see Gel electrophoresis ).
The grid size is determined by the concentration of the acrylamide. To separate larger molecules, a lower concentration of acrylamide is required than for smaller ones.
The electrophoresis is stopped as soon as the dye has passed through the gel. The protein bands in the gel are then made visible by staining ( Coomassie staining or silver staining ).
Applications
Acrylamide gels are suitable for the separation of proteins with molecular weights between 1 and 500 kDa .
There are different variants of the PAGE:
The combination of isoelectric focusing or BAC-PAGE with SDS-PAGE is called 2D gel electrophoresis . Agarose is used as an alternative matrix with significantly larger pore diameters in an agarose gel electrophoresis .
Web links
- Protein fingerprint using SDS polyacrylamide gel electrophoresis
- Discontinuous native protein gel electrophoresis
Individual evidence
- ↑ Reinhard Rüchel, Russell L. Steere, Eric F. Erbe: Transmission-electron microscopic observations of freeze-etched polyacrylamide gels. In: Journal of Chromatography A. 166, 1978, p. 563, doi : 10.1016 / S0021-9673 (00) 95641-3 .