How to perform native protein electrophoresis?

Native PAGE (Native Polyacrylamide Gel Electrophoresis) is a protein separation technique conducted under non-denaturing conditions. The purpose of native PAGE is to separate proteins while maintaining their original structure and biological activity based on charge, shape, and size. The operation of native PAGE is relatively simple, with the following steps:

1. Prepare Polyacrylamide Gel:

• Select an appropriate polyacrylamide gel concentration, generally between 5-20%. Higher concentrations are used to separate proteins with smaller molecular weights.
• Prepare the polyacrylamide gel solution according to experimental requirements, including Solution A (acrylamide, Tris chelating buffer pH 6.8 or 8.8) and Solution B (bisacrylamide).
• Mix solutions A and B, then add TEMED (N, N, N’, N’-tetramethylethylenediamine) and APS (ammonium persulfate) to initiate polymerization.
• Pour the mixture into the electrophoresis tank equipped with glass plates and wait for the gel to solidify while avoiding bubble formation.

2. Sample Preparation and Loading

• Dissolve the protein sample to be tested in a non-denaturing sample buffer (without SDS and reducing agents) to maintain the protein's native conformation.
• Select appropriate electrophoresis markers according to experimental requirements, such as dye precursors or molecular weight standards.
• Set up the electrophoresis gradient above the gel tank and load the samples and markers.

3. Perform Electrophoresis:

• Use an appropriate electrophoresis buffer, such as Tris-Glycine buffer.
• Ensure the polyacrylamide gel is submerged in the electrophoresis buffer and remove any bubbles.
• Set the appropriate voltage and electrophoresis time. Typically, the initial voltage is set to 100V, and once the samples enter the separating gel, increase the voltage to 150-200V.

4. Post-Electrophoresis Processing:

• Remove the polyacrylamide gel from the electrophoresis tank and stain it to visualize protein distribution using metal-sensitive stains such as Coomassie Blue or silver stain.
• After fixation, cutting, and extraction, proteins can undergo further experiments like mass spectrometry or immunoblotting.

Please note that data from native PAGE only provides information on protein electrophoretic mobility and cannot directly give the accurate molecular weight of proteins. Depending on experimental needs, other protein separation techniques like 2D electrophoresis, isoelectric focusing, or stacking electrophoresis can be considered.

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