Native Polyacrylamide Gel Electrophoresis for Proteins
Native polyacrylamide gel electrophoresis (Native PAGE) is an electrophoretic technique for separating proteins under non-denaturing conditions. Unlike SDS-PAGE, Native PAGE preserves the native three-dimensional structure and biological function of proteins.
Principle
Native PAGE operates by applying an electric field to induce the migration of proteins through a polyacrylamide gel. The mobility of proteins in the gel is determined by their net charge, molecular size, and conformation. Under non-denaturing conditions, proteins maintain their native three-dimensional structures. As a result, different proteins exhibit distinct migration patterns due to variations in charge and shape.
Procedures
1. Sample Preparation
Protein samples are first prepared by extracting proteins from cells or tissues. A suitable buffer is used to stabilize proteins and preserve their biological activity.
2. Gel Preparation
The polyacrylamide gel is prepared according to the required separation conditions.
3. Electrophoresis
Protein samples are loaded into the wells of the gel, and electrophoresis is conducted under an applied electric field. Proteins migrate within the gel based on their charge and molecular size.
4. Staining and Visualization
Following electrophoresis, proteins are stained with specific dyes and subsequently visualized to detect protein bands.
Advantages
1. Preservation of the Native Protein State
Native PAGE enables the separation of proteins in their native state, which is crucial for certain biochemical and biophysical analyses.
2. Maintenance of Biological Function
Since proteins remain non-denatured, their biological function is preserved.
3. Resolution of Complex Protein Assemblies
This technique is useful for separating complex protein assemblies and their subunits.
Disadvantages
A primary limitation of Native PAGE is its inability to directly determine protein molecular weight, as protein mobility is influenced not only by molecular size but also by charge and conformation. Additionally, because proteins retain their native structures under non-denaturing conditions, certain proteins may oligomerize or form high-order complexes, which can affect their migration behavior in the gel.
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