The Novex® NuPAGE® SDS-PAGE Gel System is a revolutionary high-performance polyacrylamide gel electrophoresis system that simulates the denaturing conditions of the traditional Laemmli system (Tris-glycine SDS-PAGE gels) without using SDS detergent. NuPAGE® SDS-PAGE Gels use a unique buffer formulation that maintains a low operating pH during electrophoresis. This eliminates the “smiles” and poor resolution seen with Tris-glycine SDS-PAGE gels.
The Novex® NuPAGE® SDS-PAGE Gel System provides:
*from date of manufacture
NuPAGE® SDS-PAGE Gel Electrophoresis System Components
- NuPAGE® Bis-Tris Pre-Cast Gels for separating small to mid-size molecular weight proteins
- NuPAGE® Tris-Acetate Pre-Cast Gels for separating large molecular weight proteins
- NuPAGE® LDS Sample Buffer
- NuPAGE® Reducing Agent
- NuPAGE® Antioxidant
- NuPAGE® MES SDS or MOPS SDS Running Buffer for NuPAGE® Bis-Tris Gels
- NuPAGE® Tris-Acetate SDS Running Buffer for NuPAGE® Tris-Acetate Gels
- NuPAGE® Transfer Buffer for blotting of NuPAGE® Pre-Cast Gels
NuPAGE® SDS-PAGE Gels — Neutral-pH Gel Buffer System
|Figure 1. Protein separation using (A) a Novex® NuPAGE® gel and (B) a traditional Tris-glycine gel. The samples listed below were run on (A) a NuPAGE® 4–12% Bis-Tris Gel in MES-SDS Running Buffer or (B) another manufacturer’s 4–20% Tris-glycine gel. Note the high resolution of the sample bands in the NuPAGE® protein gel. The poorly resolved “fuzzy” bands seen in the other manufacturer’s gel are a result of reoxidation of some disulfide bonds within the sample, leading to slight changes in migration rates. Lanes 1, 6, 7, 10: Mark12™ Unstained Protein Standard; lane 2: High Protein Load (4 proteins, 6 µg/protein); lane 3: Broad Range Standard; lanes 4, 5: E. coli extract; lane 8: SeeBlue® Pre-stained Protein Standard; lane 9: MultiMark Multi-colored Protein Standard.|
NuPAGE® SDS-PAGE Gels — Bis-Tris and Tris-Acetate Buffer Systems
NuPAGE® protein gels are available in two buffer systems: NuPAGE® Bis-Tris Gels and NuPAGE® Tris-Acetate Gels. The NuPAGE® Bis-Tris Gels provide superior separation of small to medium-sized proteins and employ a neutral-pH environment that minimizes protein modification. The NuPAGE® Tris-Acetate Gels are designed to provide optimal separation for large proteins at pH 8.1.
NuPAGE® Bis-Tris Gels are used in combination with either NuPAGE® MES or MOPS buffer. The NuPAGE® MES Buffer is recommended for resolving small proteins and for samples containing a broad range of protein sizes. The NuPAGE® MOPS Buffer is best for resolving medium-sized proteins.
NuPAGE® Tris-Acetate Gels are used with NuPAGE® Tris-Acetate SDS Running Buffer to resolve high molecular weight proteins (36–400 kDa) under denaturing conditions, or with Novex® Tris-Glycine Native Running Buffer to resolve high molecular weight proteins under non-denaturing (native) conditions. Figure 2 shows the migration patterns of Sharp Protein Standards on NuPAGE® Bis-Tris Gels using MOPS and MES buffers, and NuPAGE® Tris-Acetate Gels using NuPAGE® Tris-Acetate SDS Running Buffer.
Figure 2. Migration patterns of Novex® Sharp Protein Standards (prestained and unstained) on NuPAGE® Bis-Tris Gels and NuPAGE® Tris-Acetate Gels. For optimal results, protein bands should migrate within the yellow shaded areas.
NuPAGE® SDS-PAGE Gels Maintain Sample Integrity
Sample preparation is a crucial step in successful protein analysis. The pH of the traditional Laemmli-style sample buffer changes from 6.8 to 5.2 when heated at 100°C. This lower pH is known to induce aspartyl-prolyl (Asp-Pro) peptide bond cleavage, which leads to protein degradation. By maintaining a >7.0 pH environment, the NuPAGE® LDS Sample Buffer preserves protein integrity by minimizing this Asp-Pro cleavage. Figure 3 demonstrates that sample integrity is maintained throughout electrophoresis with the NuPAGE® system; protein degradation is seen in samples prepared with Laemmli (Tris-glycine) sample buffer. The NuPAGE® Antioxidant running buffer additive greatly minimizes protein oxidation during electrophoresis and keeps reduced protein bands sharp and clear.
Figure 3. Integrity of samples is maintained throughout electrophoresis with the Novex® NuPAGE® SDS-PAGE Gel System (left), compared to samples prepared with Laemmli (Tris-glycine) sample buffer (right).
NuPAGE® SDS-PAGE Gels:
Increased Gel Stability
The NuPAGE® protein gels are discontinuous SDS-PAGE gel systems that operate in the same way as the traditional Tris-glycine system but are prepared at a lower pH. This neutral-pH environment allows a much longer shelf life (1 year for NuPAGE® Bis-Tris Gels and 8 months for NuPAGE® Tris-Acetate Gels).
Efficient Western Blot Transfer
The NuPAGE® Transfer Buffer maintains neutral pH and prevents reoxidation of reduced samples during protein transfer to a membrane. This avoids sample modifications that can occur at the alkaline pH of traditional transfer buffers and maintains sample antigenicity. NuPAGE® Bis-Tris Gels are able to separate proteins using lower acrylamide concentrations than are required for Tris-glycine gels. This more open gel matrix allows for more efficient transfer of proteins to membranes during western blotting.
Best All-Around Performance
For routine protein separation, use the NuPAGE® SDS-PAGE Gel System for superior performance. In addition, the high-resolution separation and reduced protein modification and degradation properties make this system suitable for even the most demanding applications. Because the same protein separation can be achieved at lower acrylamide concentrations using NuPAGE® Bis-Tris Gels compared to Tris-glycine gels, western blot transfers are also more efficient.
The green features of NuPAGE® SDS-PAGE Gel products include: