How to Perform Native Protein Electrophoresis
Native Polyacrylamide Gel Electrophoresis (Native PAGE) is a protein separation technique performed under non-denaturing conditions. The goal of Native PAGE is to separate proteins based on their charge, shape, and size while preserving their native structure and biological activity. The operation is relatively simple, and the steps are as follows:
1. Prepare Polyacrylamide Gel
Choose the appropriate concentration of polyacrylamide gel, typically between 5-20%. Higher concentrations separate smaller proteins. Prepare the polyacrylamide gel solution, including Solution A (acrylamide, Tris buffer pH 6.8 or 8.8) and Solution B (bisacrylamide). Mix A and B, add TEMED (N,N,N’,N’-Tetramethylethylenediamine) and APS (Ammonium Persulfate) to initiate polymerization. Pour the mixture into the electrophoresis chamber with glass plates, allowing the gel to solidify while avoiding air bubbles.
2. Sample Preparation and Loading
Dissolve the protein sample in a non-denaturing sample buffer (without SDS or reducing agents) to maintain the protein’s natural conformation. Select appropriate electrophoresis markers, such as dye precursors or molecular weight standards, depending on experimental needs. Set up the electrophoresis gradient above the gel chamber, loading the samples and markers.
3. Perform Electrophoresis
Use an appropriate electrophoresis buffer, such as Tris-Glycine buffer. Ensure the polyacrylamide gel is immersed in the buffer and remove any air bubbles. Set the appropriate voltage and electrophoresis time. Typically, the initial voltage is set at 100V, and once the sample enters the gel, the voltage is increased to 150-200V.
4. Post-Electrophoresis Processing
Remove the polyacrylamide gel from the electrophoresis chamber and stain it to visualize protein distribution. Use metal-sensitive dyes (e.g., Coomassie Blue) or silver staining. After staining, protein bands can be cut, excised, and extracted for subsequent experiments such as mass spectrometry or Western blotting.
Note that Native PAGE data provides electrophoretic mobility information of the proteins, but does not directly give the exact molecular weight. Depending on the experimental needs, other protein separation techniques, such as 2D electrophoresis or stacked electrophoresis, can also be considered.
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