NuPAGE Bis-Tris Gels

High sensitivity, resolution, and confidence with NuPAGE Bis-Tris Gels

Invitrogen NuPAGE Bis-Tris Gels are high-performance gels that offer optimized chemistry for high sensitivity applications. 

Neutral-pH buffers in NuPAGE Bis-Tris Gels deliver sharp straight bands. During separation, NuPAGE Bis-Tris Gels operate close to pH 7. In the Laemmli system, the gel is run at basic pH (pH ~9.5). At high pH the residual unpolymerized acrylamide can react with cysteine and lysine residues of the proteins being separated. This may affect downstream analysis such as western blot transfer. At the neutral pH of NuPAGE Bis-Tris Gels, these reactions take place several orders of magnitude more slowly than at the basic pH of tris-glycine gels, resulting in sharper bands and better resolution.

Protein separation comparing NuPAGE Bis-Tris Gel and traditional tris-glycine gel
Protein separation comparing NuPAGE Bis-Tris Gel and traditional tris-glycine gel. The samples listed below were run on (A) a NuPAGE 4–12% Bis-Tris Gel in Invitrogen NuPAGE 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: Invitrogen Mark12 Unstained Standard; lane 2: high protein load (4 proteins, 6 µg/protein); lane 3: Broad Range Standard; lanes 4, 5: E. coli extract; lane 8: E-PAGE SeeBlue Pre-stained Standard; lane 9: Invitrogen MultiMark Multi-Colored Protein Standard.

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 Invitrogen NuPAGE LDS Sample Buffer preserves protein integrity by minimizing this Asp-Pro cleavage. As seen below, the sample integrity is maintained throughout electrophoresis with the Invitrogen NuPAGE system; protein degradation is seen in samples prepared with Laemmli (tris-glycine) sample buffer. The Invitrogen NuPAGE Antioxidant running buffer additive greatly minimizes protein oxidation during electrophoresis and keeps reduced protein bands sharp and clear.

High sample integrity
High sample integrity. Integrity of samples is maintained throughout electrophoresis with the NuPAGE SDS-PAGE Gel System (left), compared to samples prepared with Laemmli (tris-glycine) sample buffer (right).
Migration chart for NuPAGE Bis-Tris Gels