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Separate proteins according to the net charge, size and shape of their native structure using native PAGE gels. Invitrogen offers three different gel chemistries that provide sensitive, high-resolution analysis of native proteins.
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.
Gel system | Novex Tris-Glycine | NuPAGE Tris-Acetate | NativePAGE Bis-Tris |
Operating pH range | 8.3-9.5 | 7.2-8.5 | ~7.5 |
Features | Traditional Laemmle system | Better resolution of larger molecular weight proteins | Resolution of all proteins in the gel by molecular weight regardless of isoelectric point (pI) using G-250 dye |
Use when |
|
| Studying membrane proteins or hydrophobic proteins, or when you want to separate by molecular weight |
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.
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):
Recommended sample buffer | Tris-Glycine Native Sample Buffer |
Recommended running buffer | Tris-Glycine Native Running Buffer |
Recommended transfer buffer | Tris-Glycine Transfer Buffer |
Available polyacrylamide concentrations | 6%, 8%, 10%, 12%, 14%, 16%, 4–12%, 4–20%, 8–16%, 10–20% |
Available gel sizes | Mini: 8 cm x 8 cm (1.0 mm thick) Midi: 8 cm x 13 cm (1.0 mm thick) |
For use with (equipment) mini gels | Mini Gel Tank or XCell SureLock Mini-Cell |
For use with (equipment) midi gels | XCell4 SureLock Midi-Cell or Bio-Rad Criterion (with adapters only) |
Well type | Mini: 1D, wedge well format (load up to 60 µL per well) Midi: 1D |
Shelf life | Up to 12 months |
Storage conditions | 2-8 C |
Recommended sample buffer | Tris-Glycine Native Sample Buffer |
Recommended running buffer | Tris-Glycine Native Running Buffer |
Recommended transfer buffer | NuPAGE Transfer Buffer |
Available polyacrylamide concentrations | 7%*, 3–8% |
Available gel sizes | Mini: 8 cm x 8 cm (1 or 1.5 mm thick) Midi: 8 cm x 13 cm (1.0 mm thick) |
For use with (equipment) mini gels | Mini Gel Tank or XCell SureLock Mini-Cell |
For use with (equipment) midi gels | XCell4 SureLock Midi-Cell or Bio-Rad Criterion (with adapters only) |
Well type | Mini: 1D Midi: 1D |
Shelf life | Up to 8 months |
Storage conditions | Room temperature |
*7% polyacrylamide is only available in the mini size
Recommended sample buffer | NativePAGE Sample Buffer, NativePAGE 5% G-250 Sample Additive |
Recommended running buffer | NativePAGE Running Buffer, NativePAGE Cathode Buffer Additive |
Recommended transfer buffer | NuPAGE Transfer Buffer |
Recommended transfer membrane | PVDF (do not use nitrocellulose, as it tightly binds G-250 dye) |
Available polyacrylamide concentrations | 3–12%, 4–16% |
Available gel sizes | Mini: 8 cm x 8 cm (1.0 mm thick) |
For use with (equipment) mini gels | Mini Gel Tank or XCell SureLock Mini-Cell |
Well type | Mini: 1D |
Shelf life | Up to 6 months |
Storage conditions | Room temperature |
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.
Gel system | Novex Tris-Glycine | NuPAGE Tris-Acetate | NativePAGE Bis-Tris |
Operating pH range | 8.3-9.5 | 7.2-8.5 | ~7.5 |
Features | Traditional Laemmle system | Better resolution of larger molecular weight proteins | Resolution of all proteins in the gel by molecular weight regardless of isoelectric point (pI) using G-250 dye |
Use when |
|
| Studying membrane proteins or hydrophobic proteins, or when you want to separate by molecular weight |
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.
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):
Recommended sample buffer | Tris-Glycine Native Sample Buffer |
Recommended running buffer | Tris-Glycine Native Running Buffer |
Recommended transfer buffer | Tris-Glycine Transfer Buffer |
Available polyacrylamide concentrations | 6%, 8%, 10%, 12%, 14%, 16%, 4–12%, 4–20%, 8–16%, 10–20% |
Available gel sizes | Mini: 8 cm x 8 cm (1.0 mm thick) Midi: 8 cm x 13 cm (1.0 mm thick) |
For use with (equipment) mini gels | Mini Gel Tank or XCell SureLock Mini-Cell |
For use with (equipment) midi gels | XCell4 SureLock Midi-Cell or Bio-Rad Criterion (with adapters only) |
Well type | Mini: 1D, wedge well format (load up to 60 µL per well) Midi: 1D |
Shelf life | Up to 12 months |
Storage conditions | 2-8 C |
Recommended sample buffer | Tris-Glycine Native Sample Buffer |
Recommended running buffer | Tris-Glycine Native Running Buffer |
Recommended transfer buffer | NuPAGE Transfer Buffer |
Available polyacrylamide concentrations | 7%*, 3–8% |
Available gel sizes | Mini: 8 cm x 8 cm (1 or 1.5 mm thick) Midi: 8 cm x 13 cm (1.0 mm thick) |
For use with (equipment) mini gels | Mini Gel Tank or XCell SureLock Mini-Cell |
For use with (equipment) midi gels | XCell4 SureLock Midi-Cell or Bio-Rad Criterion (with adapters only) |
Well type | Mini: 1D Midi: 1D |
Shelf life | Up to 8 months |
Storage conditions | Room temperature |
*7% polyacrylamide is only available in the mini size
Recommended sample buffer | NativePAGE Sample Buffer, NativePAGE 5% G-250 Sample Additive |
Recommended running buffer | NativePAGE Running Buffer, NativePAGE Cathode Buffer Additive |
Recommended transfer buffer | NuPAGE Transfer Buffer |
Recommended transfer membrane | PVDF (do not use nitrocellulose, as it tightly binds G-250 dye) |
Available polyacrylamide concentrations | 3–12%, 4–16% |
Available gel sizes | Mini: 8 cm x 8 cm (1.0 mm thick) |
For use with (equipment) mini gels | Mini Gel Tank or XCell SureLock Mini-Cell |
Well type | Mini: 1D |
Shelf life | Up to 6 months |
Storage conditions | Room temperature |
For Research Use Only. Not for use in diagnostic procedures.