Polyacrylamide gel electrophoresis (PAGE)

Polyacrylamide, as a medium for gel electrophoresis was introduced by Ornstein. It has the advantage of being a synthetic gel, which is highly reproducible. Moreover, the pore size can be controlled by varying the proportions of acrylamide and the  crosslinking agent, bisacrylamide  Polyacrylamide can be used as a direct replacement for starch, though it is less conveniently used than  starch  in the horizontal  slab format because polymerization of polyacrylamide  is inhibited by oxygen, so it must be cast into a sealed mould.

Disc electrophoresis

Polyacrylamide was first introduced concurrently  with a new electrophoretic method  called  “disc  electrophoresis”.  This  was  first conducted in glass tubes, in which the  separated  protein  bands constituted a series of discs.  The method  also  embodied discontinuities  in the  buffer and gels used and so it  may  be described as discontinuous  electrophoresis, abbreviated to disc.  electrophoresis.  Today, because of the better cooling and the  fact that  comparison  of different  samples  is  facilitated,  disc. electrophoresis is generally conducted in vertical gel slabs.  With this  layout, the sample is applied to one end of the gel slab and so, unlike with a horizontal slab gel lay-out, only anions or cations, but not both, can be analyzed at one time.  The original method of Ornstein5, is an anionic system (i.e. anions are analyzed).

In an anionic system,  there  is usually a common  buffer cation,  e.g. Trisí throughout.  In  the  buffer compartments,  a buffer with an anionic

component having  a pH-dependent electrophoretic  mobility is used, e.g. glycine-. Above its pI, the anodic mobility of glycine increases with pH as the proportion of glycine- ions increases.

In the  gels, an anionic component  having  a high,  pH-independent mobility, e.g. Cl^-, is used.

Two gels are used; a large pore stacking gel and a smaller pore  running gel.  At the outset, both gels contain Tris-HCl buffer, but at different pH values.  The  buffer in the stacking gel is of lower pH than  that  in the gel. A schematic view of the apparatus at the start of an electrophoresis run is shown in Fig. 1.  The samples are mixed with sucrose or glycerol to increase their density and are layered directly under the electrode buffer. Upon application of the electrical field, a sharp interface develops between the high mobility so-called “leading ion”, i.e. Cl^-, and the less mobile so-called “trailing ion”, i.e. glycine, according to Kohlrausch’s regulating functions. In order to visualize the interface between the leading and trailing ions a small amount of a dye,  such as bromophenol blue, may be added to the samples or to the upper electrode solution.

Figure 1. Experimental set-up for discontinuous PAGE.

As the interface moves downward, the protein molecules with mobilities intermediate between Cl- and glycine, will be swept up and concentrated into a very thin band.  Within this band, individual proteins will become stacked in  order of their mobilities,  with  those of highest mobility  immediately next to the Cl- ions. During the later parts of this so-called stacking phase, therefore, all of the proteins will move downwards at the same speed, i.e. this could be called an isotachophoresis stage (iso = “the same”, “tach” = speed).  The concentrated band of protein has a higher density than the surrounding buffer and the system would be convectively unstable were it not stabilized by the stacking gel.

The voltage gradient  (-dV/dx) within the separating part of the apparatus will not be uniform but will have a stepwise decrement at the interface between the leading and trailing ions.  If proteins  are additionally present, the step will be comprised  of a number of smaller sub-steps, corresponding to interfaces between the different proteins.

Figure 2.  The voltage profile during the stacking phase of disc gel electrophoresis.

Arrows in Fig. 2 indicate the direction of movement of the  interface and its associated voltage  discontinuity.  The  voltage  gradient is  steeper behind the interface than in front.  This  follows from the  fact that all the ions are moving at the  same speed.  For ions  of a low mobility  to  move as fast as ions of a high mobility,  they  must  be  in  a  steeper  voltage gradient. Any protein which falls behind the interface, say by diffusion. will find itself in an area with a steep  voltage  gradient and will thus be accelerated towards the interface,  Conversely  any protein  which diffuses ahead of the  interface  will enter  an  area with a shallow gradient and will slow down and be overtaken  by the  moving  interface.  In  this  way  the proteins are focused into thin layers in the interface.

When the interface reaches the junction between the  stacking and running gels, two things happen.  Firstly the  pH  increases,  resulting  in  an increase in the mobility of the glycine trailing ion as a larger proportion will exist in the  glycine- form at the higher pH.  Secondly, the proteins encounter the sieving effect of the  small pore running gel.  Together these result in the leading ion/trailing ion interface overtaking the stack of proteins,  which are  left  to  separate  in  a  linear  voltage  gradient according to their respective mobilities in the running gel.

The bromophenol blue dye remains with the  interface,  making its progress easily visible.  When the interface reaches near the end of the gel the  electric  field can  be switched off,  in the  sure  knowledge  that  no proteins will have migrated further than the interface and that all will still be in the  gel.  The  separated proteins  can  be visualized by  staining,  for example with Coomassie blue, and destaining.

A number of disc PAGE systems have been described by Jovin.

Isotachophoresis

Although it is not a PAGE system, it is convenient to discus isotachophoresis at this point because it is conceptually related to the stacking phase of disc electrophoresis.

In the later stages of the  stacking phase  of disc electrophoresis,  the proteins  are  stacked on top of one  another  in  very  thin  layers  (Fig.  3). If the  electrophoresis  is  conducted  in  a tube,  then  the  thin  layers  will  be in the form of discs.

Figure 3.  Proteins stacked as thin layers in the stacking phase of disc gel electrophoresis.

The different proteins are in fact separated from one another, although adjacent bands are touching,  but this  separation  is not  useful as the bands are so thin that it is impossible to distinguish between them. However, some improvement in the  situation could be achieved by making the tube  much thinner,  so that  a given amount  of protein  would occupy a greater length.

Ultimately, if the separation was carried out in a capillary tube, the bands would occupy  a greater length  in the  capillary tube.  In  this situation, it is possible to  distinguish the different bands.  At each interface there is a step change in the voltage gradient, which corresponds to a change in resistance due to  the  fact that  the  proteins have different mobilities.  This  change in resistance at  each  interface corresponds to a change in heat production and this  can be detected with a thermocouple detector.  The output is a sharp peak as each interface passes the detector.

In this way the number of proteins  present can be determined, but they cannot be identified.  If one protein was missing, the others  would simply close up and one fewer interface would be detected  but it  would be difficult to  determine  which protein  was missing.  This  problem  can  be overcome by adding a mixture  of “ampholytes”  to  the  protein  sample. Ampholytes are synthetic  polyamino-polycarboxylic acids, in which the amino and carboxyl groups are randomly added to a carbon chain backbone - they are sold under a number of trade  names,  e.g. Ampholine, Servalyte, Bio-lyte. The result is a mixture of molecules with a range of pI values, resulting in a range of electrophoretic mobilities.

Because of their  range of mobilities, there  will always be some ampholyte molecules with mobilities intermediate between those of the different proteins  and in isotachophoresis  these  will serve to  space the protein molecules a distance apart from one another, and so are known as “spacers”. The  separated  protein  molecules  can  be  detected  by UV absorption. Ampholytes will be revisited later in a discussion of isoelectric focusing.

Isotachophoresis is discussed here largely as a conceptual  development of the  stacking  phase  of disc  electrophoresis.  In  fact  it  is  a  technique which is not  much  used.  However,  the  concept  of conducting electrophoresis in a capillary tube has been highly successful and capillary electrophoresis has become a versatile and popular analytical technique, which can be applied to  proteins  and other  ions.  An  advantage  of the capillary system is that a capillary tube at once  controls  convection  and gives excellent cooling so that  high voltages can be used, giving rapid separations. A down-side to  capillary  electrophoresis  is that  the apparatus tends to be expensive.

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 .