Native polyacrylamide gel electrophoresis

Native PAGE uses the same discontinuous chloride and glycine ion fronts as SDS-PAGE to form moving boundaries that stacks and then separates polypeptides by charge to mass ratio. Proteins are prepared in a non-reducing non-denaturing sample buffer, which maintains the proteins’ secondary structure and native charge density. Therefore you can easily see multiple bands from the camshot of your native PAGE gel if your target protein is in polymerized forms in your sample. In native PAGE electrophoresis most proteins have an acidic or slightly basic pH(isoelectric point) (~3–8) and migrate towards the negative pole. If your protein's pl is larger than 8,9, then reverse the anode and run the native PAGE gel.

Steps for experiment : 

1. Prepare appropriate amount of separating gel in a small beaker, then add specific volume of AP and TEMED and gently swirl the beaker to ensure a sufficient mixing. Pour the gel solution into the gap between the glass plates of gel casting but don't fully fill. Fill the rest space with water (isopropanol alternatively). Allow 20-30min for a complete gelation.

2. You can prepare the stacking gel solution while the separating gel is gelating. Prepare appropriate amount of stacking gel in a beaker and mix with 10% AP and 1% TEMED. Pour out the water in the first step and pipet the stacking gel solution into the gap and insert the comb. Allow 20-30min to let it gelate.

3. Mix your sample with sample buffer. Do not heat your sample.

4. Load the sample mixture and set an appropriate voltage to run the electrophoresis.

5. Stain as you would for standard Coomassie-blue protocol or proceed to a immuno-blotting procedure (western-blot).