Preparation of 10x Tris-Glycine Electrotransfer Buffer for Western Blot


The 10x Tris-Glycine Transfer Buffer can be used to transfer proteins from polyacrylamide gel to nitrocellulose or PVDF membranes by electroblotting. The 10x buffer is prepared by dissolving 30.3 g Tris base and 144.1 g Glycine in deionized water to a final volume of 800 ml. Note that 10x Tris-Glycine Transfer Buffer does not contain methanol. Methanol is added while preparing 1x buffer.

The 10x buffer can be stored for a long time at room temperature. This buffer is suitable for transferring proteins from Bio-Rad TGX gel to nitrocellulose or PVDF membrane. The same buffer can also be bought from Bio-Rad (10x Tris/Glycine Buffer for Western Blots and Native Gels #1610734).

A 1x buffer is prepared by diluting 100 ml of 10x buffer in the mix that contains 200 ml Methanol and 700 ml deionized water. So the final 1x transfer buffer contains 25 mM Tris, 192 mM glycine, and 20% Methanol. 


Reagents and solutions
Tris base (Molecular Weight: 121.14) (e.g., Sigma-Aldrich #T1503)
Glycine (Molecular Weight: 75.07
) (e.g., Sigma-Aldrich #G8898)
Deionized water

* Tris base (CAS Number: 77-86-1) and Trizma® base (CAS Number: 77-86-1) are the same chemicals.

Equipment and disposables
Beaker of suitable size (use a 2 L beaker to prepare 1 L solution)
Magnetic stirrer and beads
Weighing balance
Measuring cylinder

Running buffer composition (10x)
0.31 M Tris
2.39 M Glycine
pH 8.3

Running buffer composition (1x)
25 mM Tris
192 mM Glycine
20% Methanol
pH 8.3

Preparation of 1L of 10x SDS-PAGE Running Buffer


Step 1: Weigh out 30.3 g Tris base and 144 g Glycine and transfer to a 2L beaker.

Step 2: Add 600 ml of deionized water and stir until all reagents dissolve completely.

Step 3: Adjust the final volume to 800 ml using deionized water.
Transfer the solution from the beaker to a 1L measuring cylinder and add deionized water to the measuring cylinder to bring the final volume to 800 ml.

Note: The pH of the solution should be 8.3 without any adjustment.

The solution can be stored at room temperature for a year.

Preparation of 1x solution

To prepare 1L of 1x solution containing 20% methanol, take 700 ml deionized water in a measuring cylinder, and add 200 ml Methanol and 100 ml 10x Tris-Glycine Transfer Buffer. Mix and use for transfer.

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