Application of Tris buffer in protein electrophoresis and nucleic acid agarose electrophoresis

Details:

 Tris buffer  is a component of protein electrophoresis and Western blotting. Most SDS-PAGE gel, electrophoretic buffer and imprinted buffer were buffered by Tris.

Application of Tris buffer in protein electrophoresis

Most SDS gels use discontinuous Tris buffer systems. Concentrated gels have macropores, so larger peptides can be easily migrated through, usually under pH6.7 – 6.8. At this pH, ionized chloride ions migrate rapidly, increasing the pH behind them and generating a voltage gradient with a low conductivity region, which causes glycine (from the running buffer) to ionize and migrate behind the chloride front. Most peptides in the sample are negatively charged due to the binding of SDS and migrate between chloride and glycine to form a narrow band, thus "stacking". Once the stack reaches the separation gel, it is at a higher pH value (usually pH value 8.7-8.8), and the increase of glycine ionization will accelerate and exceed the peptide segment. In addition, the separation gel with smaller pore size began to produce screening effect, resulting in the separation of peptide segments according to size.

Most Western blot protocols use Tris buffer with low ionic strength for protein transfer. The transfer time depends on the type of imprinting device and the peptide size range of interest.

Application of Tris buffer in nucleic acid agarose electrophoresis

Tris buffer is widely used in DNA agarose electrophoresis. The two main buffers are tbe (Tris borate / EDTA) and Tae (Tris acetate / EDTA). Although there are some differences in the resolution of different forms of DNA and their mobility during electrophoresis, these Tris buffers can usually be used interchangeably. Borate ions in tbe inhibit many enzymes, so some enzyme mediated downstream operations may not work if some type of DNA purification is not carried out after electrophoresis. Therefore, Tae buffer is favored in daily use in most DNA laboratories.

Making Tris buffer

Tris buffer is a good choice for most biological systems because its pKa value at 25 ° C is about 8.1, making it an effective buffer in the pH 7-9 range. This pH range is applicable to most biological processes. Tris powder is also cheaper and more durable than more professional buffers such as HEPES.

There are two methods to prepare Tris buffer solution. One is to prepare Tris base and Tris HCl solution with the required concentration, then add an aliquot of one solution (usually Tris HCl) to another (usually Tris base) solution, and monitor the pH value until the correct pH value is obtained. In practice, this is rarely done.

The common way to make most common Tris buffers is to start with tris bases only. Dissolve an appropriate amount of Tris powder in water, adjust the pH value with HCl, and then prepare the buffer into the required volume. Assuming that there is no overshoot when adjusting the pH value, the method will not change the ionic strength. However, the ionic strength will change after overshoot and readjustment with NaOH, or after Tris HCl and pH adjustment with NaOH. Depending on the intended use of the tris buffer, such changes may or may not be important.

When making Tris buffer, it is important to use Tris compatible electrodes when adjusting pH. When used with tris buffer, the single junction Ag / AgCl electrode will show instability because silver will gradually precipitate and block the electrode. The use of double junction or calomel reference electrodes ensures accurate pH measurement in Tris buffer.

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