Native PAGE Gels

Three different gel chemistry systems are available for native PAGE separation (Tris-Glycine, Tris-Acetate and NativePAGE Bis-Tris). There is no universal gel chemistry system ideal for the electrophoresis of all proteins in their native state. Protein stability, resolution and isoelectric point are important considerations for the buffer selection.

In native PAGE, proteins are separated according to the net charge, size, and shape of their native structure. Electrophoretic migration occurs because most proteins carry a net negative charge in alkaline running buffers. The higher the negative charge density (more charges per molecule mass), the faster a protein will tend to migrate. At the same time, the frictional force of the gel matrix creates a sieving effect, regulating the movement of proteins according to their size and three-dimensional shape. Small proteins face only a small frictional force, while larger proteins face a larger frictional force. Thus, native PAGE separates proteins based upon both their charge, mass and structure.

Because no denaturants are used in native PAGE, subunit interactions within a multimeric protein are generally retained and information can be gained about the quaternary structure. In addition, some proteins retain their enzymatic activity (function) following separation by native PAGE. Thus, this technique may be used for preparation of purified, active proteins.

How NativePAGE Bis-Tris Gels work

In standard SDS-PAGE, the charge-shift molecule is SDS. The SDS denatures proteins and binds to them, which confers a net negative charge; this allows the proteins to migrate in one direction towards the anode. The SDS is present in the sample buffer and running buffer. NativePAGE Bis-Tris Gels use Coomassie G-250 to bind to proteins and confers a net negative charge while maintaining the proteins in their native state without protein denaturation. The G-250 is present in the cathode buffer to provide a continuous flow of G-250 into the gel and is added to samples containing non-ionic detergent prior to loading the samples onto the gel. The gels do not contain any G-250. This system, based on the blue native polyacrylamide gel electrophoresis (BN PAGE) technique developed by Schägger and von Jagow, overcomes the limitations of traditional native gel electrophoresis by providing a near-neutral operating pH and detergent compatibility.

PVDF is the recommended blotting membrane for western blotting with NativePAGE Gels. Nitrocellulose is not compatible for blotting NativePAGE Gels since the nitrocellulose membrane binds the Coomassie G-250 dye very tightly and is not compatible with alcohol-containing solutions used to destain the membrane and fix the proteins.

The binding of G-250 to proteins offers the following advantages resulting in high-resolution native electrophoresis (Schägger, 2001):

• Proteins with basic isoelectric points (pI) normally have a net positive charge, G-250 converts the proteins to a net negative charge, allowing the proteins to migrate in one direction towards the anode.
• Membrane proteins and proteins with significant surface-exposed hydrophobic area are less prone to aggregation as G-250 binds nonspecifically to hydrophobic sites converting them to negatively charged sites.