2.2.3 Native Polyacrylamide Gel Electrophoresis (Native-PAGE)
Native Polyacrylamide Gel Electrophoresis (Native-PAGE)
Native Polyacrylamide Gel Electrophoresis (Native-PAGE), also known as activity electrophoresis, is a technique that performs polyacrylamide gel electrophoresis on proteins while maintaining their biological activity, without adding denaturants such as SDS (Sodium Dodecyl Sulfate) and mercaptoethanol. It is commonly used for enzyme identification, isozyme analysis, and purification.
In native polyacrylamide gel electrophoresis without SDS, biomacromolecules can retain their natural shape and charge during the electrophoresis process. Their separation is based on the differences in electrophoretic mobility and the molecular sieve effect of the gel, thus achieving high resolution. Particularly, after electrophoretic separation, biomacromolecules such as proteins and enzymes can still maintain their biological activity, which is of great significance for the identification of macromolecules.
The operation of native polyacrylamide gel electrophoresis is basically the same as that of denaturing SDS-PAGE. The only difference is that the preparation of native polyacrylamide gel and the electrophoresis buffer must not contain denaturants like SDS.
When conducting native gel electrophoresis for general proteins, it is necessary to distinguish between basic proteins and acidic proteins.
For separating basic proteins, a low-pH gel system should be used.
For separating acidic proteins, a high-pH gel system should be used.
Acidic proteins usually adopt a buffer system with a pH of 8.8 in native gel electrophoresis. Under this condition, the proteins carry a negative charge and migrate towards the anode.
In contrast, the electrophoresis of basic proteins is typically carried out in a slightly acidic environment, where the proteins carry a positive charge. In this case, it is necessary to reverse the positions of the cathode and anode to achieve the electrophoretic separation of basic proteins.
Reagent Formulation for Native-PAGE
1. Separating Gel
Reagent Volume
Double-distilled Water 6.6 ml
30% Acrylamide Solution 8.0 ml
1.5 mol/L Tris (pH 8.8) 5.0 ml
10% Ammonium Persulfate (APS) Solution (W/V) 200 μl
TEMED (Tetramethylethylenediamine) 15 μl
2. Stacking Gel
Reagent Volume
Double-distilled Water 6.8 ml
30% Acrylamide Solution 1.7 ml
1 mol/L Tris (pH 6.8) 1.25 ml
10% Ammonium Persulfate (APS) Solution (W/V) 100 μl
TEMED (Tetramethylethylenediamine) 10 μl
