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NuPAGE® - Reach for the Best

NuPAGE®Bis-Tris Gels and NuPAGE®Buffers
  • NuPAGE® Gels yield the best resolution and most consistent results.
  • NuPAGE® Gels have a long shelf-life - at least 8 months!
  • NuPAGE® Gels transfer more efficiently than Tris-Glycine Gels.
  • Minimal Protein Modification - Ideal for Protein Sequencing & Mass Spectrometry
  • Simple, Effective Sample Reduction System
  • More Efficient Western Transfers
  • Fast Run Times: 35-50 minutes
The NuPAGE® System: Advantages of the Neutral pH/gel Buffer System

The NuPAGE® Pre-Cast Gel System is a revolutionary polyacrylamide gel system for high performance gel electrophoresis. It consists of NuPAGE® Bis-Tris Pre-Cast Gels (for small to mid-size molecular weight proteins), NuPAGE® Tris-Acetate Gels (for larger proteins) and specially optimized buffers. The unique formulation and low operating pH during electrophoresis offer significant advantages over other gel systems. Advantages of a Lower pH While there are subtle differences between the chemistries of Bis-Tris and Tris-Acetate gels, both provide a much lower pH environment than traditional SDS-PAGE systems. The advantages of lower pH include sharper band resolution, longer shelf-life, and higher accuracy of results. For many years, the Laemmli system has been the standard method used to perform SDS-PAGE.Laemmli-type gels (upon which the NOVEX® Tris-Glycine pre-cast gel chemistry is based) are useful for a broad range of protein separations. The Tricine gel system was later developed to extend the separation range to small peptides. The Laemmli system has certainly proved its worth, but it is subject to some potential problems:

  • The gel is cast at pH 8.7. At this pH, polyacrylamide under-goes gradual hydrolysis, which eventually causes band distortion and/or loss of resolution. Consequently, the shelf life of a Tris-Glycine gel is typically only 1-2 months.
  • The pH of the separating region of the gel is about 9.5 during electrophoresis. At this pH, proteins are potentially subjected to chemical modifications such as deamination and alkylation. This may affect subsequent analyses of proteins fol-lowing the run.
  • The redox state of the gel is not well controlled. This means that reduced disulfides are more prone to reoxidation, giving rise to diminished band sharpness and transfer efficiency, particularly for cysteine-containing proteins.
  • Proteins are subject to cleavage at asp-pro bonds when heated in the Laemmli sample buffer . Also, reduction of disulfides is inefficient in the Laemmli sample buffer at pH 6.8. Either can lead to artifact bands or poor resolution.

Increased Gel Stability

The NuPAGE® line of gels eliminates the problems associated with the traditional Laemmli system. Both the NuPAGE® Bis-Tris and the new NuPAGE® Tris-Acetate gels are discontinuous SDS-PAGE systems that operate in the same way as the traditional Tris-Glycine system, but are cast at a lower pH (pH 6.4 in Bis-Tris gels and pH 7.0 in Tris-Acetate gels). This results in:

  • Dramatically improved gel stability . This means a much longer shelf life (guaranteed 1 year for NuPAGE® Bis-Tris gels and 8 months for NuPAGE® Tris-Acetate gels).
  • Better protein stability during the run. The pH of the separat-ing region is 7.0 for the Bis-Tris gels and 8.1 for the Tris-Acetate gels. Better protein stability is particularly important for low abundance proteins or critical post-gel analysis such as peptide sequencing and mass spectrometry analysis.

Optimized Buffers
The NuPAGE® LDS Sample Preparation Buffer (pH 8.4), used in both the NuPAGE® Bis-Tris and NuPAGE® Tris-Acetate Systems, has been optimized to:

  • Reliably provide complete reduction of disulfides under mild conditions (70°C, 10 minutes)
  • Eliminate protein cleavage during sample preparation
The NuPAGE® Antioxidant running buffer additive, used in reducing conditions and blotting protocols in both the Bis-Tris and Tris-Acetate systems:
  • Greatly minimizes protein oxidation during electrophoresis and keeps reduced protein bands sharp and clear
  • Contributes to better transfer efficiency by eliminating inter-protein disulfide formation

NuPAGE® Bis-Tris Gels: High-Performance Gels for SDS-PAGE

NuPAGE® Bis-Tris Gels provide the best separation and resolution of small to medium sized proteins by utilizing a neutral pH environment which minimizes protein modifications. These high-performance gels also have a one-year shelf life when stored at +4 to 25°C, eliminating the waste of throwing out expired gels. NuPAGE® Bis-Tris Gels are a great choice for protein sequencing, mass spectrometry, and any other assays where protein integrity is crucial. The NuPAGE® system also provides the most efficient means for transferring proteins to membranes for subsequent analysis.

The NuPAGE® System is based upon a Bis-Tris-HCl buffered (pH 6.4) polyacrylamide gel, with a separating gel that operates at pH 7.0. While NuPAGE® Bis-Tris Gels do not contain SDS, they are formulated for denaturing gel electrophoresis applications only. NuPAGE® Bis-Tris Gels are available in three acrylamide concentrations: 10%, 4-12%, and 12%. By combining any of these three gel types with the NuPAGE® MES or MOPS Buffers, six separation
ranges can be obtained.

Contents & Storage

The minimum order for NuPAGE® Gels is 10. NuPAGE® Bis-Tris Gels may be stored at +4 to 25°C. Shelf life is 12 months.

Recommended Buffers:

  • Running Buffers: NuPAGE® MES or NuPAGE® MOPS Running Buffers should be used with NuPAGE® Bis-Tris Gels. Each buffer produces a different migration pattern when used with the same gel. By combining these two buffers with the three NuPAGE® Bis-Tris Gel types, 6 different migration patterns can be obtained.
  • Sample Prep Buffers: Use of the NuPAGE® LDS Sample Buffer and NuPAGE® Reducing Agent will ensure complete and consistent sample reduction. The NuPAGE® Antioxidant is added to the upper (cathode) buffer chamber to prevent reduced proteins from reoxidizing during electrophoresis. For non-reducing sample preparations, the NuPAGE® LDS Sample Buffer should be used without the Reducing Agent and Antioxidant.
  • Transfer Buffers: The NuPAGE® Transfer Buffer was designed specifically for blotting NuPAGE® gels. The NuPAGE® Antioxidant is added to the transfer buffer for enhanced blotting results with reduced proteins.

Gel Migration

The shaded area of the chart represents the area of maximum resolution. Select a gel where your molecule of interest will migrate to the shaded area of the gel. For example, proteins ranging from 21kDa to 36kDa would be resolved best on a 10% NuPAGE® Bis-Tris Gel with MOPS Running Buffer. The bands for the protein gels correspond to the migration of our Mark12 Wide Range Standard under denaturing conditions.

Table 1 - Conversion: Current NOVEX SDS-PAGE Gels to NuPAGE

Tris-Glycine Tricine Bis-Tris Gel + Running Buffer
18%, 16%, 14% 16% 10% + MES SDS
12%, 10%, 8% 10% 10% + MOPS SDS
4-20%, 10-27% 10-20% 4-12% + MES SDS
8-16%, 4-12% -- 4-12% + MOPS SDS
  • NuPAGE® Bis-Tris 4-12% and 10% Gels, 1.5 mm, 10 Well are used in protein separations, under denaturing conditions. The gels are 1.5mm thick. Each well will hold a maximum sample volume of 37µl. The gel cassette is 10 cm x 10 cm.
    NuPAGE® Bis-Tris 4-12% and 10% Gels, 1.5 mm, 2D Well are used in 2D protein separations, under denaturing conditions, following isoelectric focusing. The gels are 1.5mm thick. Each well will hold a maximum sample volume of 600µl. The gel cassette is 10 cm x 10 cm.
    NuPAGE® Bis-Tris Gels are lot qualified by measurement of migration of protein standards over time.
    NuPAGE® Bis-Tris Gels are sold 10 per box.
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