assay protocol

Native PAGE Tips

Some details in native PAGE can be easily mistaken or forgotten. Here lists the tips to pay attention to.

1. Different buffers for different pl of your proteins:

High-pH gel system for separation of acidic protein. Generally if your protein is acidic, it's better to ajust your buffer system to pH 8.0-9.0. In this condition acidic protein is negatively charged and can migerate to the positive anode in the gel.

Low-pH gel system for separation of alkaline protein. Normally electrophoresis is performed in a slightly acidic buffer invironment. To separate alkaline proteins, the cathode and anode need to be inverted before native gel running.

2. Loading:

The ionic tensity of your sample should not be higher than 0.1mmol/L, otherwise it would cause a deformation of the electrophoretic bands.

A centrifuge before loading to prevent loading the pellet.

Generally, a marker is not needed in native PAGE gel running. Buy specific native PAGE markers if you need one.

3. Gel running:

A pre-running is prefered. (30-60min)

Take care of the voltage. Typically, it should not be lower than 100V and not higher than 200V.