NuPAGE Tris-Acetate Gels

NuPAGE Tris-Acetate Gels are designed to give optimal separation of large molecular weight proteins during gel electrophoresis. In comparison to traditional Tris-glycine SDS-PAGE gels, NuPAGE Tris-Acetate Gels have an environment of pH 8.1, which minimizes protein modifications and results in sharper bands.

 Features of NuPAGE Tris-Acetate Gels include:

• High resolution—gels offer optimal separation of high molecular weight proteins
• Better protein integrity—sample preparation process has been optimized to help preserve your proteins
• Better transfer of large proteins—the specialized gel buffer and lower polyacrylamide concentration near the top of NuPAGE Tris-Acetate gradient gels allows larger proteins to transfer more efficiently
• Longer shelf life—gels can be stored for at least 8 months

NuPAGE Tris-Acetate Gels do not contain SDS and so can be used to separate proteins in native or denatured form. For denatured proteins, we recommend using LDS sample buffer and tris-acetate SDS running buffer. For native proteins, traditional Tris-glycine native sample buffer and a Tris-glycine native running buffer should be used.

The NuPAGE Tris-Acetate Gels use a discontinuous buffer system involving three ions:

• Acetate (–) is from the gel buffer and serves as a leading ion due to its high affinity to the anode relative to other anions in the system. The gel buffer ions are tris (+) and acetate (–) (pH 7.0).
• Tricine (–) is from the running buffer and serves as the trailing ion. The running buffer ions are tris (+), tricine (–) and dodecylsulfate (pH 8.3).
• Tris (+) is the common ion present in the gel buffer and running buffer. The Tris-acetate system also operates at a significantly lower operating pH of 8.1 during electrophoresis. 

Better 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 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.

Better transfer of high molecular weight proteins using NuPAGE Tris-Acetate gels

The low concentration of polyacrylamide concentration near the top of NuPAGE Tris-Acetate gels allows proteins of larger molecular weights to escape the gel matrix during protein transfer and results in more sensitive western blot detection compared to gels with higher polyacrylamide concentrations near the top of the gel. A comparison is shown below using a NuPAGE 3-8% Tris-Acetate midi gel, 12+2 well run against a Bio-Rad 7.5% TGX midi gel, 12+2 well.

Decreasing amounts of HEK 293 cell lysate prepared in RIPA lysis buffer (48 – 0.5 µg total protein) were denatured in the respective manufacturer’s sample buffer and subjected to electrophoresis using manufacturer instructions.

Western blots using NuPAGE 3-8% Tris-Acetate midi gels generate sharp bands at high protein and RIPA lysis buffer loads while larger proteins fail to transfer from Bio-Rad 7.5% TGX midi gels. NuPAGE 3-8% Tris-Acetate midi gel, 12+2 well, 12+2 well, was loaded with decreasing total protein amount of HEK293 lysate, subjected to electrophoresis in a SureLock Tandem Midi Gel Tank and transferred onto a 0.45 µm PVDF membrane using the SureLock Tandem Blot Module and the Bio-Rad 7.5 % TGX midi gel, 12+2 well, was subjected to electrophoresis in a Criterion Midi Cell Tank and transferred onto a 0.45 µm PVDF membrane using the Criterion Blotter. For these gels, 10 µL of HiMark Prestained Protein Ladder was used as the protein standard. Both membranes were probed simultaneously for two protein targets (BRCA2 and HDAC1) using chemiluminescent immunodetection.  BRCA2 was not detected in the Bio-Rad TGX midi gel, which indicates inefficient transfer of this large protein from the Bio-Rad gel. NuPAGE 3-8% Tris-Acetate precast protein gels were designed for electrophoresis and western blotting of large proteins and large proteins tend to transfer more efficiently from the lower acrylamide concentration near the top of the 3-8% acrylamide gradient gel.

For immunodetection, membranes were blocked for 1 hour in 1X Blocker FL Fluorescent Blocking Buffer.  For chemiluminescent detection, the membranes were probed overnight with a mixture of primary antibodies diluted in blocking solution: Rabbit-anti BRCA2 (1:500) and Rabbit anti-HDAC1 (1:5,000,000) followed by an incubation with secondary antibody in 1X Blocker FL: Donkey anti-Rabbit HRP (1:5,000) for 1 hour.  Membranes were incubated for 5 minutes with SuperSignal West Dura Extended Duration Substrate and imaged on an iBright Imaging System for the same amount of time and the same image adjustment settings.