2.2.4 Tris-Acetate Gel

Reliable Separation and Transfer of High-Molecular-Weight (HMW) Proteins

Reliable separation and transfer of high-molecular-weight (HMW) proteins is a common challenge faced by researchers in the life sciences.
Selecting the correct gel is a key factor for the successful transfer of HMW proteins.
The popular universal gels are 4% to 20% Tris-glycine gels, which can effectively separate a variety of proteins.
However, HMW proteins will be compressed into a narrow region at the top of the gel.
For HMW proteins, better choices are Tris-acetate gels or low-percentage non-gradient Tris-glycine or Bis-Tris gels.
When specifically analyzing HMW proteins, Tris-acetate gels can be used to achieve optimal transfer results.
Compared with Bis-Tris or Tris-glycine gels, Tris-acetate gels maintain a neutral pH and separate HMW proteins with higher resolution.
By comparing the effects of different gel systems and gradients on the separation of HMW proteins, it can be seen that 3‒8% Tris-acetate gels provide the most ideal separation effect and resolution for HMW proteins.
This improvement in resolution leads to higher transfer efficiency and greater sensitivity.
△ Click to enlarge the image
Tris-acetate gels yield the best results for separating HMW proteins.
(A) Ideal separation results are obtained in the yellow shaded area.
(B) Better transfer is observed when using Tris-acetate gels on 4‒20% Tris-glycine gels:
9 ng is detected on the Tris-acetate gel, while 620 ng is detected on the Tris-glycine gradient gel.
Reagent Formulation for Tris-Acetate Polyacrylamide Gradient Gels
3M Tris-acetate pH 7.0 Gel Buffer (15X, 100ml):
Weigh 36.33g of Tris base and dissolve it in 90ml of ultrapure water. Mix thoroughly and adjust the pH to 7.0 using acetic acid. Make up the volume to 100ml and store at 4°C.
LDS Sample Buffer (4X):
Combine 4ml of 2.5 M Tris-HCl (pH 8.5), 0.8g of LDS, 0.006g of EDTA, 5ml of 80% glycerol, 0.75ml of 1% CBB G250 solution, and 0.25ml of 1% phenol red solution. Mix thoroughly and store at room temperature.
40% Acrylamide Solution:
A 37.5:1 mixture of acrylamide to bis-acrylamide. Store at 4°C.
APS: 10% Solution.
APS: 10% Solution.
TEMED: Store at 4°C.
TEMED: Store at 4°C.
DTT (1,4-Dithiothreitol): 1ml aqueous solution, store at -20°C.
DTT (1,4-Dithiothreitol): 1ml aqueous solution, store at -20°C.
Running Buffer (1X):
Dissolve 8.95g of N-tris(hydroxymethyl)methylglycine, 6.06g of Tris base, 1g of SDS, and 0.25g of sodium bisulfite in ultrapure water. Make up the volume to 1L.
The pH of this solution is approximately 8.24; store at 4°C. Prepare all solutions using ultrapure water (Milli-Q quality) and analytical-grade reagents.
Gel Casting
△ Click to enlarge the image
The figure above is a schematic diagram illustrating how to prepare a 3‒15% gradient Tris-acetate gel using a gradient former.
Pour the 3% and 15% polyacrylamide solutions [prepared from a 40% acrylamide (37.5:1 acrylamide/bis-acrylamide mixture) stock solution] into the left and right compartments of the gradient former, respectively, at the same time. Immediately after completing the pouring of the gradient gel, add the 3% solution, and then allow the gradient gel to polymerize.