NuPAGE Tris-Acetate Gels

Superior resolution of large molecular weight proteins

Invitrogen NuPAGE Tris-Acetate Gels simulate the denaturing or the native conditions of the traditional Laemmli system. A unique buffer formulation helps to maintain a low operating pH during electrophoresis, resulting in superior resolution of proteins compared to traditional tris-glycine SDS-PAGE gels.

Reliable separation and transfer of high molecular weight (HMW) proteins is a common challenge for life science researchers. Choosing the right gel is a key factor in the successful transfer of HMW proteins. A popular general-use gel is a 4–20% tris-glycine gel, which can effectively separate a mixed range of proteins. However, HMW proteins will be compressed into a narrow region at the top of the gel. A better option for HMW proteins is a tris-acetate gel or a low percentage non-gradient tris-glycine or bis-tris gel. When specially targeting HMW proteins, optimal transfer can be achieved with a tris-acetate gel.

Tris-acetate gels maintain a neutral pH and separate HMW proteins with higher resolution than bis-tris or tris-glycine gels. Comparison of HMW protein separation using different gel chemistries and gradients shows best separation and resolution of HMW proteins using a 3–8% tris-acetate gel. This increased resolution leads to increased transfer efficiencies and higher sensitivity.

Tris-acetate gels enable the best separation of HMW proteins
Tris-acetate gels enable the best separation of HMW proteins. (A) Optimal results are obtained in the yellow shaded areas. (B) Better transfer is seen using the tris-acetate gel over a 4–20% tris-glycine gel: 9 ng is visualized on the tris-acetate gel versus 620 ng visualized on the tris-glycine gradient gel.

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. 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.

 NuPAGE SDS-PAGE Gel System compared to samples prepared with Laemmli (tris-glycine) sample buffer
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 charts for NuPAGE Tris-Acetate Gels