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Study Notes: Buffer Systems

The choice of the buffer system, and the interaction of this buffer system with the sample as the sample moves through the gel matrix, is what achieves the separation of the proteins into discrete bands on the gel. There are basically three kinds of buffer systems.

Homogenous Buffer System
The identity and concentration of buffer components are the same in the gel and both tanks. This is used for most forms of DNA and RNA electrophoresis but this is generally not used for protein electrophoresis, where a multiphasic buffer system is used.

In this homogeneous system, the buffer protects the samples as well as carrying the current. One common problem that may arise is where the ionic strength of the gel is different to either of the tanks, often due to an error in preparation. In this case a ‘salt wave’ moves through the gel during running, leading to localised distortions to the band pattern.

Multiphasic Buffer Systems
As the name implies this system uses differing buffers and is used for SDS-PAGE (often called the Laemmli system). Above the separating gel is a lower percentage acrylamide gel with a different buffer. This gel acts to ‘stack’ the protein bands into sharp zones prior to separation in the higher strength separating gel.

Buffers used are:

  • Stacking Gel - Tris-HCl, pH 6.8
  • Separating Gel - Tris-HCl, pH 8.8 at a higher concentration
  • Tanks - Tris-glycine, pH 8.8.

These gel and buffer discontinuities produce sharp separations amongst sample components. At the beginning of the electrophoresis run the buffer concentrations are as listed above but this changes as chloride and glycinate ions migrate down the stacking gel; the chloride ions migrate faster (leading ions) than the glycinate ions (trailing ions). This leads to the development of a voltage gradient between the two ion fronts and the sample molecules are carried along in this thin front leading to ‘stacking’ of the sample molecules into thin discrete layers in order of electrophoretic mobility (ease of migration).

Once the ion front moves into the separating gel the conditions become similar to an homogeneous gel and molecular sieving of the sample molecules occurs in this higher strength separating gel.

Glycine ions are at the very top of the tank, chloride ions in the middle.

At the start of the run, chloride ions (green) are in the gels and glycine ions (orange) are in the running buffer tanks.

 

Some chloride ions have moved into the bottom tank.

With the establishment of the ion boundary in the stacking gel (blue wavy lines) the proteins are stacked into a thin layer in this boundary.

   

Glycine ions and chloride ions have moved down the tank.

Once in the separating gel, electrophoresis proceeds based on size of the sample molecules.

Isotachophoresis
Uses a non-sieving low percentage gel (essentially one large stacking gel) and is used for difficult samples such as very small peptides (small proteins). The stacking of sample molecules occurs via ion front effects, as with the multiphasic system, but of course the strong resolving power of a separating gel is absent.

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