Equilibrium isoelectric focussing in acrylamide gel slabs—Reduction of cathodic drift
✍ Scribed by Leon Hunter
- Book ID
- 102982940
- Publisher
- Elsevier Science
- Year
- 1978
- Tongue
- English
- Weight
- 892 KB
- Volume
- 89
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
The technique of isoelectric focussing has proved to be an invaluable adjunct to traditional electrophoretic procedures, differing from the latter in that the various proteins in the sample move under an applied voltage to their isoelectric points in a preestablished pH gradient. The final focussing effect of this procedure is in marked contrast to the progressive protein band diffusion observed with conventional electrophoresis.
The original procedure developed by Svensson (l-3) was carried out in liquid pH gradients stabilized by the addition of sucrose. With appropriate precautions such gradients could be maintained in stable condition for the 20 to 100 hr necessary to allow complete migration of the sample proteins to their isoelectric points.
Subsequent developments have seen the introduction of solid or gel matrix systems to support and to stabilize the pH gradient. Among the many advantages of the latter systems have been ease of operation, substantial savings in the expensive ampholytes used to form the gradient, and increased speed of protein separation.
Polyacrylamide gels are a popular medium for this type of isoelectric focussing and there are many references to their use in the form of slabs and cylinders (4-6). Cylindrical gels have been the most successful, despite the obvious advantages of slabs for multiple sample comparisons and correlation of protein bands. Use of the latter has been discouraged by many instability problems (6) associated with pH drift-to the extent that some manufacturers of both slab and cylindrical gel equipment will recommend only the latter for isoelectric focussing. Other workers (6) and manufacturers (7) have attempted to bypass the problem by describing procedures for carrying out accelerated slab runs which are completed before pH drift becomes a serious problem. In the latter case it is evident that no true equilibrium pH gradient is ever established. Therefore, it is something of a misnomer to apply the term isoelectric focussing to such a technique.
I would like to describe a simple procedure that overcomes many of the problems associated with slab gel isoelectric focussing and which results in excellent protein band separations under pH gradient conditions which are essentially stable for periods of at least 1 to 2 days.
The prime problem in gel slabs is the so-called cathodic drift (8). Although this tendency for the pH gradient to shift toward the cathode