Native PAGE Gels

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.

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Native PAGE protein separation

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
  • Need to keep the native net charge of proteins
  • Studying smaller molecular weight proteins (20- 500kDa)
  • Need to keep the native net charge of proteins
  • Studying larger molecular weight proteins (>150 kDa)
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.

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.

Available gels and recommended buffers for Native PAGE

Native gel electrophoresis using Tris-Glycine Gels

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

Native gel electrophoresis using Tris-Acetate Gels

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

Native gel electrophoresis using NativePAGE Bis-Tris Gels

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

Novex Tris-Glycine Gels

See complete list of available gels

NuPAGE Tris-Acetate Gels

See complete list of available gels

Migration of NativeMark Unstained Protein Standard under native conditions

Comparing native page migration of NativeMark Unstained Protein Standard using Tris-glycine, tris-acetate and NativePAGE gels

Native PAGE protein separation

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
  • Need to keep the native net charge of proteins
  • Studying smaller molecular weight proteins (20- 500kDa)
  • Need to keep the native net charge of proteins
  • Studying larger molecular weight proteins (>150 kDa)
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.

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.

Available gels and recommended buffers for Native PAGE

Native gel electrophoresis using Tris-Glycine Gels

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

Native gel electrophoresis using Tris-Acetate Gels

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

Native gel electrophoresis using NativePAGE Bis-Tris Gels

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

Novex Tris-Glycine Gels

See complete list of available gels

NuPAGE Tris-Acetate Gels

See complete list of available gels

Migration of NativeMark Unstained Protein Standard under native conditions

Comparing native page migration of NativeMark Unstained Protein Standard using Tris-glycine, tris-acetate and NativePAGE gels
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