Starch gel electrophoresis

Historically, starch gel electrophoresis preceded agarose electrophoresis but here the order of discussion is turned about to group mechanistically related techniques.  Starch gel electrophoresis  was introduced by Smithies in 1955.  Starch forms microreticular, thermosetting gels comprised of interlocking starch helices, cross-linked by H-bonds. The microreticular nature of starch gels introduced the phenomenon of  gel sieving  which revolutionized  electrophoresis  by greatly increasing its resolution and sensitivity.

In a microreticular gel, a protein migrating under electrophoresis  faces a greatly increased frictional resistance,  due to  the  fact that  the  proteins have to migrate through the  3-D gel network.  This  resistance  is an inverse function of the  size of the  protein,  so that  small proteins  will migrate with less friction than  larger proteins,  while proteins  larger than the exclusion limit of the gel will not be able to enter  into  the gel at all.

Ferguson has determined that  the  mobility  of a protein  in  a starch gel, as a function of the gel concentration, is described by the equation:-

Where

i.e. the mobility decreases logarithmically as the  gel concentration increases. A plot of In  u_i vs T_S gives a straight line, of slope – ^t k, known as a Ferguson plot.

The same apparatus as used for paper electrophoresis and CAM-E can be used for starch gel electrophoresis.  The gel is cast as a horizontal  slab, which is connected to the buffer reservoirs  using filter paper wicks.  The slab must not be too thick  to  prevent  excessive  heat  build-up. For extra cooling, the  slab  may  be  supported  on  a block  internally  cooled by circulating cold water.  To accommodate the samples to be separated, small slit-like wells are cast in the  gel slab, using a purpose-made mould, or cut with a scalpel.  In the latter case the sample can  be introduced  into the slit by inserting a small strip of filter paper impregnated with sample. After sample is introduced into the sample wells, the electric  field is applied across the  length  of the  gel.  Under the influence of the electric field, the  sample proteins  will move  either  towards  the  cathode  or  the anode, depending upon their charge at the  buffer pH.  Initially, they will migrate through the  buffer in the  sample  wells by free  electrophoresis. When they strike the well wall, on either the anodic  or  cathodic  side. the resistance to their migration will increase  and the  sample will be concentrated into a narrow band.

Figure 1.  Sharpening of starting zones in starch gel electrophoresis.

The resolution in electrophoresis is a function of the starting bandwidth and of the band spreading  during electrophoresis.  The latter is largely due to diffusion of the protein from its zone  of highest concentration. The structure of a microreticular gel, however, not only impedes the  progress  of proteins  undergoing  electrophoresis,  brit also limits diffusion.  The  combination  of  narrow  starting  bands  and  reduced diffusion results in the marked improvements in resolution and sensitivity of gel electrophoresis.  The  sensitivity  is  increased  since  proteins  are more easily detected the  higher their concentration  and, by the  initial concentration  of the  bands and  subsequent minimization  of diffusion, proteins present at low levels can be detected.

A down-side of SGE is that, due  to  the  variability  of starch  which  is a natural product,  results tend to vary from lab to lab and in the same lab  at different times.  This  motivated  a  search  for  a more  uniform,  synthetic gel. Nevertheless, starch gel electrophoresis is still widely used today, mainly by biologists exploring  the  taxonomic  relationships  of organisms or in plant  breeding.  An  advantage  of starch  gels is that  they  are  non-toxic and biodegradable and are thus suitable for large-scale screening.

References 

Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .

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