Principle of Disc Electrophoresis

Discontinuous electrophoresis solves two problems of protein electrophoresis: it prevents aggregation and precipitation of proteins during the entry from liquid sample into the gel matrix, and promotes the formation of well defined bands. The discontinuity is based on four parameters:

• the gel structure
• the pH value of the buffer
• the ionic strength of the buffer
• the nature of the ions in the gel and in the electrode buffer

The gel is divided into two areas: resolving and stacking gel. The resolving gel with small pores contains 0.375 mol/L Tris-HCL buffer pH 8.8, the stacking gel with large pores contains 0.125 mol/L Tris-HCL pH 6.8.

The electrode buffer contains only glycine, the gel only Cl- ions. Glycine has a pI 6.7, it has almost no net charge at pH 6.8: the pH of the stacking gel. Thus glycine has a low mobility.

At first, the proteins are separated according to the principle of isotachophoresis and form stacks in the order of their mobility ("stacking effect"). The individual zones become concentrated. Because of the large pores in the stacking gel, the mobilities are dependent on the net charge, not on the size of the molecule. Because of the relatively slow migration velocity of glycine, the samples enter the gel slowly without sudden concentrating.

The protein stack migrates - slowly and at constant speed - towards the anode, till it reaches the border to the resolving gel. The frictional resistance suddenly increases for the proteins, they migrate slower, and the zones become higher concentrated. The low molecular weight glycine is not affected by this, passes the proteins, and becomes more charged in the resolving zone; the new Cl- / glycine- front moves ahead of the proteins.

Several events now occur simultaneously:

The proteins are in a homogeneous buffer medium, destack and start to separate according to the principles of zone electrophoresis. Their mobility now depends on their charge as well as on their size. The ranking of the protein ions changes.

The pH value rises to 9.5 and because of this, the net charge of the proteins and increases.

Disc electrophoresis affords high resolution and good band definition.