NativePAGE™ Bis-Tris Gel System

Novex® NativePAGE™ Bis-Tris Gel System

The Novex®  NativePAGE™ Bis-Tris Gel System is a precast polyacrylamide mini-gel system that provides sensitive, high-resolution analysis of native proteins and protein complexes for molecular mass estimations, and assessment of purity.  It is based on the blue native polyacrylamide gel electrophoresis (BN PAGE) technique developed by Schägger and von Jagow [1–3] that  overcomes the limitations of traditional native electrophoresis by providing a near-neutral operating pH and detergent compatibility.

Life Technologies now offers Novex® NativePAGE™ Bis-Tris gels for blue-native electrophoresis of proteins and protein complexes, and NativeMark™ unstained standards for size estimation of proteins in native PAGE. Advantages of the NativePAGE™ Novex® Bis-Tris Gel System over traditional Tris-glycine gels include:

  • Wide molecular weight resolving range — from 15 kDa to 10 MDa
  • Neutral-pH separation — preserves better the native state of  protein complexes
  • Resolution of all proteins in the gel regardless of their isoelectric point (pI)
  • Higher resolution than with Tris-Glycine native electrophoresis

NativePAGE™ Bis-Tris Gel Products

How NativePAGE™ Gels Work

The blue native gel electrophoresis technique (using the NativePAGE™ Bis-Tris Gel System) uses Coomassie G-250 as a charge-shift molecule. In SDS-PAGE, the charge-shift molecule is SDS as it binds to proteins and confers a negative charge while at the same time denaturing the proteins. In blue native gel electrophoresis, the G-250 binds to proteins and confers a negative charge without denaturing. The G-250 is added to the cathode buffer in the system providing a continuous flow of dye into the gel, and is added to samples that contain non-ionic detergents before loading them onto the gels. The gels themselves do not contain G-250 so they appear as any other polyacrylamide gel before they are run.

The binding of G-250 to protein molecules provides two key benefits:

  • Proteins with basic isoelectric points that would normally have a net positive charge are converted to having a net negative charge so that they migrate in the correct direction — to the anode
  • Membrane proteins and other proteins with significant surface-exposed hydrophobic area are less prone to aggregation when G-250 binds nonspecifically to the hydrophobic sites converting them to negatively charged sites [3].

NativePAGE™ Gels — Native Separation Compared To Tris-Glycine

Samples requiring nonionic detergents for solubility are not compatible with traditional native Tris-Glycine PAGE because as the proteins migrate into the polyacrylamide gel, they leave behind the nonionic detergents. In the absence of nonionic detergent, the proteins aggregate and form vertical streaks at the top of the lane (Figure 1A). When using blue native electrophoresis, the coomassie G-250 dramatically reduces aggregation to allow the resolution of membrane protein complexes not seen in the Tris-glycine gel (Figure 1B). Bands that appear slightly green are chlorophyll-containing membrane protein complexes.

In addition, compared to the operative pH of the Tris-glycine system (pH 9.3-9.5), the lower operative pH of the NativePAGE™ gels (pH 7.5-7.7) may help to retain the native structure and/or activity of proteins sensitive to alkaline pH. The affect of the gel operative pH on enzyme activity was demonstrated with β-galactosidase using a post-electrophoretic in-gel activity assay, Figure 2 [4]. While both gel types show sharp band resolution, the NativePAGE™ gel displays greater retention of enzyme activity.

 

 
Figure 1.   Native protein gel electrophoresis was performed with 4-12% Novex® Tris-glycine (A) or 4-16% NativePAGE™ gels (B). Gels were loaded with NativeMark™ standards (lane 1), 18μg spinach chloroplast extract solubilized in 0.25%, 0.5%, 1.0%, and 2.0% dodecylmaltoside (lanes 2–5, respectively). Staining was performed with Colloidal Blue Staining Kit.

Figure 2.  β-Galactosidase was separated on NativePAGE™ Novex® or Novex® Tris-glycine gels. Both gels were equilibrated in PBS pH 7.5 for 10 minutes before incubation in chromogenic enzyme substrate solution.

NativeMark™ Unstained Protein Standard

NativeMark™ unstained protein standard is provided as ready-to-use liquid, and is compatible with multiple native gel chemistries, e.g. Tris-Glycine, Tris-Acetate and NativePAGE™ systems.  The marker offers a very wide molecular weight range of 20 kDa to over 1,200 kDa, and the 242 kDa B-phycoerythrin band is visible as a red band after electrophoresis (prior to staining) for reference.

Migration patterns on Novex® NativePAGE™ protein gel
 
Figure 3. Migration patterns of NativeMark™ Protein Standards on Novex® NativePAGE™, Tris-Glycine, and Tris-Acetate Protein Gels.
NativeMark™ Unstainted Protein Standard Figure 4. NativeMark™ Unstained Native Protein Marker on NativePAGE™ Novex®  4–16% Bis-Tris Gel.

NativePAGE® Gel Resources

Instruments & Accessories

 
Buffers & Reagents

 
Technical Resources

Application Notes

References

  1. Schägger, H., and von Jagow, G. (1991) Blue Native Electrophoresis for Isolation of Membrane Protein Complexes in Enzymatically Active Form. Anal. Biochem. 199:223-231.
  2. Schägger, H., Cramer, W. A., and von Jagow, G. (1994) Analysis of Molecular Masses and Oligomeric States of Protein Complexes by Blue Native Electrophoresis and Isolation of Membrane Protein Complexes by Two-dimensional Native Electrophoresis. Anal. Biochem. 217:220-230.
  3. Schägger, H. (2001) Blue Native Gels to Isolate Protein Complexes from Mitochondria. Meth. Cell Biol. 65:231-244.
  4. Manchenko, G. P. (1994) Handbook of Detection of Enzymes on Electrophoretic Gels, CRC Press, Boca
    Raton.

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