Disc electrophoresis

 

Discontinuous electrophoresis according to Ornstein and Davis solves two problems during the separation of proteins in gels with small pores:

 

1.  It prevents the precipitation of proteins during sample entry into the gel.

2.  It promotes the formation of well defined bands.

 

The discontinuity is based on four parameters:

 

· gel structure

· pH value of the buffer

· ionic strength of the buffer

· nature of the ions in the gel and in the electrode huffer

 

The gel matrix is divided into two areas:

 

·     the resolving gel and

·     the stacking gel.

 

Resolving gel:

small pores

0.375 mol/L TrisHCL buffer

pH 8.8

 

Stacking gel:

large pores

0.125 mol/L TrisHCL buffer.

(pH 6.8)

 

Electrode buffer:

 

Only slow terminating ions such as glycine

(Glycine has a very low net charge at the pH value of the stacking gel).

 

In the gel:

Exclusively leading ions with high mobility such as Cl^-

 

Principle :

 

The proteins are separated according to the principle of isotachophoresis and form stacks in the order of their mobility ("stacking effect").

--> The individual zones are concentrated. (The mobilities are dependent on the net charge, not on the size of the molecule)

The proteins migrate slowly and at constant speed towards the anode till it reaches the limit of the separation gel.

The frictional resistance of the proteins suddenly increases;

glycine is not affected by this, passes the proteins, and becomes more highly charged in the resolving zone; the new Cl ^- glycine front moves ahead of the proteins.

In the original work, chloride ions were used in the buffer.

Now, many people use phosphate ions in the stacking gel or in the resolving and stacking gel.

 

Several events now occur simultaneously:

· The proteins are in a homogeneous buffer medium and start to separate according to the principles of zone electrophore

· 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 increases.

 

Disc electrophoresis affords high resolution and good band definition.

 

In the example cited above, proteins with pl’s greater than pH 6.8 migrate in the direction of the cathode and are lost.

 

Another buffer system must be chosen to separate these proteins. A selection can be found in the works of Maurer and Jovin.

 

The stacking and resolving gels are only cast together just before electrophoresis because, when the complete gel is left standing for a long time the ions diffuse towards one another.