APPLICATION NOTE
NativePAGE™ Bis-Tris gels and buffers
NativePAGE™ Bis-Tris gels and buffers
for blue-native electrophoresis
Advances in proteomics research have increased the
need for better tools to investigate the properties of
newly identified proteins. Among the tools that will
assist researchers in this area is native electrophoresis.
The ability to maintain native protein conformation and
quaternary structure, coupled with the unparalleled
resolution capability of electrophoresis, makes this
technique a powerful tool for the analysis of protein–
protein interactions. Traditional native electrophoresis has
been limited in its applicability for native protein analysis
because of the high operative pH of the Tris-glycine
system that may adversely affect proteins sensitive to
high-pH conditions.
The BN PAGE technique 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 BN PAGE, 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 nonionic 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:
Additionally, traditional native electrophoresis is limited
by its incompatibility with native samples that require
a nonionic detergent for protein solubility. In 1991,
Schägger and von Jagow developed a technique called
blue-native polyacrylamide gel electrophoresis (BN
PAGE) that overcomes the limitations of traditional native
electrophoresis by providing a near-neutral operating pH
and detergent compatibility [1]. Life Technologies offers
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.
• 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 areas are less prone
to aggregation when G-250 binds nonspecifically to
the hydrophobic sites and converts them to negatively
charged sites [2]
NativePAGE™ gels improve native separations over
Tris-glycine gels
Samples requiring nonionic detergents for solubility are
not compatible with traditional Tris-glycine native PAGE
because as the proteins migrate into the polyacrylamide
gel, they leave the nonionic detergents behind. In the
absence of nonionic detergent, the proteins aggregate
near the top of the lane (Figure 1A). In blue-native
electrophoresis, Coomassie™ G-250 dramatically reduces
aggregation to allow the resolution of membrane protein
complexes not seen in the Tris-glycine gel (Figure 1B).
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 effect of the operative pH on enzyme
activity was demonstrated with β-galactosidase using a
post-electrophoretic in-gel activity assay (Figure 2) [3].
While both gel types show sharp band resolution, the
NativePAGE™ gel displays greater retention of enzyme
activity.
4-12% Tris Glycine
4-16% Blue Native
A(+) G-250 Cathode B (+) G-250 Cathode
1
2
3
4
5
1
2
3
4
A
Figure 2. Retention of activity of β-galactosidase after separation
on NativePAGE™ or Tris-glycine gels. Both gels were equilibrated
in PBS, pH 7.5, for 10 minutes before incubation in a chromogenic
enzyme substrate solution.
Two-dimensional NativePAGE™/SDS-PAGE separations
Analyses of the subunit composition of protein
complexes can be performed through two-dimensional
electrophoresis (2DE) employing NativePAGE™ gels in the
first dimension and denaturing SDS-PAGE gels in the
second dimension (Figure 3). Due to the compatibility of
NativePAGE™ gels with membrane protein complexes,
these types of 2D gels may provide an alternative to
traditional 2DE (IEF/SDS-PAGE) for the analysis of
hydrophobic membrane proteins. Many of the complexes
seen in Figure 3 as bands in the first-dimension separation
are integral membrane proteins (chloroplast photosystems
and mitochondrial oxidative phosphorylation complexes).
5
Protein–protein interaction analysis
Native electrophoresis can provide another tool for
verification of protein–protein interactions identified by
other methods. NativePAGE™ gels were used to evaluate
expression and protein complex formation from the
750kD
10MD 2.5MD
I V III
IV
II
BN PAGE
NuPAGE/MES
Figure 1. Native electrophoresis on traditional Tris-glycine vs.
NativePAGE ™ (blue-native) gels. (A) Tris-glycine 4–12% gel. (B)
4–16% NativePAGE ™ Bis-Tris gel. Lane 1: NativeMark™ standards;
lanes 2–5: 18 µg spinach chloroplast extract solubilized in 0.25%,
0.5%, 1.0%, and 2.0% dodecylmaltoside. Gels were stained with the
Colloidal Blue Staining Kit.
B
Figure 3. Bovine mitochondrial extract solubilized with 1%
dodecylmaltoside and separated on a 3–12% NativePAGE™ gel in
the first dimension (BN PAGE) and 4–12% NuPAGE® SDS-PAGE in
the second dimension (NuPAGE/MES). Staining was performed
with SYPRO® Ruby Protein Gel Stain.
Figure 4. Analysis of protein complex formation from NativePure™
expression of capTEV ™-tagged 20S proteasome subunit β4 bait
protein. Post-nuclear supernatants of untransfected control (lane
2), C-term tagged bait (lane 3), and N-term tagged bait (lane 4) were
separated on 3–12% NativePAGE ™ gels and then either (A) stained
for protein or (B) western-blotted for streptavidin-AP detection.
Signal detected above background in lane 3 shows the tagged 20S
proteasome complex.
NativePure™ system (Figure 4). Protein complex formation
was verified only for the C-term tagged bait protein (Figure
4, lane 3), so the N-term tagged sample was not subjected
to affinity purification.
The proteins calmodulin and calcium/calmodulindependent protein kinase 1 were identified as interacting
on yeast protein arrays [4]. An in-solution binding
reaction with purified proteins was performed to verify
the interaction with an electrophoretic mobility shift
assay (Figure 5). A band with a larger apparent molecular
weight was detected with increasing signal intensity that
correlated with the increase in calmodulin protein. No
band-shift signal was detected in control lanes upon
overexposure (data not shown).
NativeMark™ molecular weight markers
for all native gels
The NativeMark™ Unstained Protein Standard is provided
as a ready-to-use liquid solution and is compatible with
multiple native gel chemistries (Figure 6). It 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.
Figure 5. Mobility shift assay verification of protein interaction
between calmodulin (CaM) and calcium/calmodulin-dependent
protein kinase 1 (Cmk1). The indicated amounts of protein were
mixed in 1X NativePAGE ™ sample loading buffer supplemented with
5 mM CaCl2, and incubated at 37°C for 1 hour before separation on
a 4–16% NativePAGE ™ gel. Chemiluminescent immunodetection was
performed with 1:500 Novex® rabbit anti-calmodulin.
Tris-Acetate Gels
3-8%
1236
1048
Tris-Glycine Gels
1236
1048
720
480
720
4-12%
6%
7%
1048
720
1236
1048
720
480
480
242
8-16%
242
146
242
242
146
146
1048
1048
720
720
480
720
242
480
480
242
146
146
66
66
66
20
4-16%
1048
146
480
Blue Native Gels
4-20%
3-12%
1236
1048
242
720
146
480
66
20
242
146
20
20
66
Figure 6. NativeMark™ Unstained Protein Standard (5 μL) on
NuPAGE® Tris-acetate and Tris-glycine gels run with Tris-glycine
native running buffer, or NativePAGE™ gels run with NativePAGE™
running buffer. All were 10-well gels and stained with the Colloidal
Blue Staining Kit.
Ordering information
Product
NativePAGE™ 4–16% Bis-Tris Mini Gels, 1.0 mm, 10 well
Quantity
Cat. No.
10 pack
BN1002BOX
NativePAGE 3–12% Bis-Tris Mini Gels, 1.0 mm, 10 well
10 pack
BN1001BOX
NuPAGE® 4–12% Bis-Tris Mini Gels, 1.0 mm, 10 well
10 pack
NP0321BOX
NuPAGE® 4–12% Bis-Tris Mini Gels, 1.0 mm, 10 well
2 pack
NP0321PK2
NuPAGE 3–8% Tris-Acetate Mini Gels, 1.0 mm, 10 well
10 pack
EA0375BOX
NuPAGE 3–8% Tris-Acetate Mini Gels, 1.0 mm, 10 well
2 pack
EA0375PK2
™
®
®
Novex 4–12% Tris-Glycine Mini Gels, 1.0 mm, 10 well
10 pack
EC6035BOX
NativeMark™ Unstained Standard
5 x 50 µL
LC0725
Colloidal Blue Staining Kit
1 kit
LC6025
SYPRO Ruby Protein Gel Stain
1L
S-12000
SYPRO Ruby Protein Gel Stain
200 mL
S-12001
Tris-Glycine Native Running Buffer (10X)
500 mL
LC2672
®
®
®
Summary
Blue-native electrophoresis performed with NativePAGE™ Bis-Tris gels provides a valuable tool for analyzing membrane
protein complexes, protein complex subunit structure, protein–protein interactions, enzyme activity, and size estimation of
native proteins.
References
1.Schägger H, 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 (2001) Blue-native gels to isolate protein complexes from mitochondria. Meth Cell Biol 65:231–244.
3.Manchenko GP (1994) Handbook of Detection of Enzymes on Electrophoretic Gels. CRC Press Inc. p. 228.
4.Zhu H, Bilgin M, Bangham R et al. (2001) Global analysis of protein activities using proteome chips. Science 293:2101–2105.
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