Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis is a commonly used electrophoresis technique that uses polyacrylamide gel as a supporting medium. It is often used to separate oligonucleotides and proteins. It is often used as anionic detergent and can act as a solubilizing agent and texturizing agent. The role of stacking is often mentioned when talking about the function of concentrating gel. When the concentration is relatively small, the pore size will be relatively large. Let us learn more about this aspect below.

Introduction

Principle of action: Polyacrylamide gel has a network structure and has a molecular sieve effect. There are two forms: native-PAGE and SDS-PAGE. In native-PAGE, proteins can remain intact during electrophoresis and gradually separate in a gradient according to the molecular weight, shape and charge of the protein.

SDS-PAGE can separate proteins based solely on the molecular weight of their subunits. The technology was first established by Shapiro in 1967. They found that after adding ionic detergents and strong reducing agents (SDS, sodium dodecyl sulfate) to the sample medium and acrylamide gel, the electrophoretic mobility of protein subunits mainly depends on the size of the subunit molecular weight (charge factors can be ignored).

effect

SDS is an anionic detergent. As a denaturant and solubilizing agent, it can break the hydrogen bonds within and between molecules, cause the molecules to unfold, and destroy the secondary and tertiary structures of protein molecules. Strong reducing agents such as mercaptoethanol and dithiothreitol can break the disulfide bonds between cysteine ​​residues. After adding reducing agents and SDS to the sample and gel, the molecules are depolymerized into polypeptide chains. The depolymerized amino acid side chains combine with SDS to form protein-SDS micelles. The negative charge they carry greatly exceeds the original charge of the protein, thus eliminating the charge and structural differences between different molecules.

SDS-PAGE generally uses a discontinuous buffer system, which can have a higher resolution compared to a continuous buffer system.

The function of the concentrated gel is stacking. The gel concentration is small and the pore size is large. When a relatively dilute sample is added to the concentrated gel, it is concentrated to a narrow zone through the migration effect of the large-pore gel. When TRIS/HCl buffer is selected as the sample solution and stacking gel, TRIS/glycine is selected as the electrode solution. After electrophoresis begins, HCl dissociates into chloride ions and glycine dissociates into a small amount of glycine ions. Proteins are negatively charged, so they move toward the positive electrode together, with chloride ions moving fastest, glycine ions moving slowest, and proteins in the middle. At the beginning of electrophoresis, the mobility of chloride ions is the largest, exceeding that of proteins, thus forming a low conductivity area at the back. The electric field strength is inversely proportional to the low conductivity area, thus generating a higher electric field strength, causing proteins and glycine ions to move rapidly, forming a stable interface, causing proteins to aggregate near the mobile interface and condense into an intermediate layer.

In this identification method, the mobility of a protein depends mainly on its relative molecular mass and has nothing to do with its charge and molecular shape.