NuPAGE Tris-Acetate Gels

NuPAGE Tris-Acetate gels offer increased resolution and higher transfer efficiency of high molecular weight proteins than Tris-Glycine and Bis-Tris gels. In addition, the neutral pH environment helps preserve protein integrity, helping improve the separation and detection of high molecular weight proteins.

• High resolution—optimal separation of high molecular weight proteins
• Great protein integrity—sample preparation process has been optimized to help preserve your proteins
• Excellent 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
• High-capacity wells—WedgeWellformat wells are wedge-shaped wells that double the well sample loading capacity and make it easy to load your sample (no specialized pipette tips required), available in mini and midi gel format

*Not all percentages are available in every well type

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.

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Reliable separation and transfer of high molecular weight (HMW) proteins is a common challenge for life science researchers. 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. Tris-acetate gels maintain neutral pH (8.1) and separate HMW proteins with higher resolution than Bis-Tris or Tris-glycine gels. This increased resolution, paired with the easier protein movement afforded by low percentage polyacrylamide, leads to increased transfer efficiencies and higher sensitivity. In addition, the neutral pH environment helps minimize protein modification and results in sharper bands.

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.

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.

Figure 2. 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.

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• NuPAGE Technical Guide
• NuPAGE Tris-Acetate Gel Mini Gel Protocol
• NuPAGE Tris-Acetate Gel Midi Gel Protocol
• Mini Gel Tank Manual
• SureLock Tandem Midi Gel Tank USER GUIDE
• XCell4 SureLock Manual
• Protein Gel Electrophoresis Handbook
• Protein Transfer Technical Handbook