Isoelectric focusing

Of the  separation  methods  based upon  gross physical  properties  such as charge or size etc.,  isoelectric focusing, is one  of the  most discriminating. Only methods based upon some biological property, which requires a subtle  stereospecific  relationship,  are  more discriminating. Sometimes IEF can almost be too discriminating because multiple bands can be obtained from what is essentially the  same glycoprotein species, with only small differences in glycosylation   a phenomenon known  as  “micro -heterogeneity”.  A downside of  IEF  is that it is a relatively expensive, because of the cost of the ampholytes required.

Proteins are  ampholytes  having a pH-dependent net charge, which is positive at pH values below the proteins  PI and negative at pH values above the PI.

Figure 1.  Schematic view of isoelectric focusing.

If proteins  are  distributed  throughout  a  solution  in  a  pH  gradient,  then upon application of an electrical potential  across the gradient, with the anode at the low pH end, molecules in the  low pH zone  (which will be positively charged) will migrate to the cathode.  Their migration will take them through zones of increasing pH, as a consequence of which they will gradually lose their positive  charge and their rate  of migration  will  slow

down. Conversely, molecules in the high pH zone will be negatively charged and will consequently migrate, through zones  of decreasing pH, towards the anode.  When  each  protein  reaches  a  position  where  the  pH is equal to its pI, it will lose all of its charge and its migration will cease. After a sufficient time, therefore,  the respective molecules will in consequence become “focused” at their isoelectric points. In this way, a mixture of proteins can be separated, as each will focus at a characteristic pI, Furthermore, the pI values can readily be measured in this way.  In practice, three difficulties must be overcome:

•  A stable, uniform pH gradient must be established and maintained.

•  The system must be stabilized against disturbances due to convection.

•  A system must be devised for the measurement of the  pH gradient and for determination of the positions of the focused bands.

1- Establishing a pH gradient

If a pure  ampholyte,  such  as  a  protein,  is  added  to  pure  water,  the water will acquire a pH equal to  the  isoionic point  of the  ampholyte which for most practical  purposes  is the  same  as the  pI  of the ampholyte18. So, a stack of ampholytes of increasing pI, arranged one on top of the other, would constitute a pH gradient. Electrophoretic mobility is also a function of pI and, as has been outlined in the  discussion of isotachophoresis  ,  it is possible to  electrophoretically  stack  ampholytes  in  order  of their mobilities. However, in isotachophoresis a buffer is present to control the pH.  If there  were  no  buffer  present,  except  the  ampholytes,  then  in arranging themselves  in order of mobility they  would simultaneously generate a pH gradient, the pH at each point  corresponding to  the  pI of the ampholyte at that point.  Since each ampholyte would finally be at its pI, where it has no net charge, there  should theoretically  be no  net movement of the pH gradient.

If the  ampholytes  making  up the  pH  gradient were  proteins,  the gradient would have a few steps (as many as there are  proteins),  but these steps would tend  to  be quite large  (Fig.  2).  However,  if synthetic, randomly substituted, polyamino-polycarboxylic acid ampholytes were used,  then  there  would be a very  large number of very  small steps, which in effect gives a smooth  pH gradient.  A protein  introduced into such a gradient will cause a plateau to be formed  at  its pI,  the  length being proportional to the amount of protein (Fig. 3).

Figure 2. A pH gradient constructed from a stack of seven proteins.

Figure 3.  A pH gradient constructed from randomly synthesized polyamino-polycarboxylic acid ampholytes (and containing one protein).

The anode solution is acidic and the cathode solution is basic. Consequently, ampholyte  molecules immediately in contact  with the anode solution will be positively charged and those  in contact  with the cathode solution will be negatively charged.  At zero time the  pH distribution across the apparatus will be  low at the  anode  end and high  at the cathode end, but with a central plateau, corresponding to the pH of the sample plus mixed ampholytes  (Fig. 4).  When the  electrical potential is applied across the electrodes, ampholytes will move to the anode or cathode, depending upon their charge, until they  reach  a pH at which they will have  zero  charge.  Each  ampholyte  species will establish the pH at its pI and so with time the  ampholytes  will arrange themselves into an order of charge and in doing so will establish a smooth  pH gradient (Fig.  4).

Figure 4. Time course of the establishment of a pH gradient in IEF.

Sample proteins added into this gradient will participate  as ampholyte species and each will focus at its particular pI,  introducing  a small  plateau in the gradient.  Generally,  it  is  desirable  that  samples  are  added  in  a  way that avoids their exposure to the extreme pH values near the electrode solutions. This consideration has made open, flat bed systems popular as the pH  gradient  can  be  established  and  the  samples  can  subsequently  be applied at a position away from the electrodes.

One of the reasons  for the  discriminating power of IEF is that  the principle of its operation  intrinsically counteracts  the  effects of diffusion, which in other methods is responsible for the  broadening of bands with time.  Any  protein  which  diffuses  out  of a  focused  band  will enter a region of different  pH where it will acquire a charge.  In consequence it will immediately experience  an electrophoretic  force tending to  move  it back into  the  focused band.  In this way the  band is kept focused.

The steepness of the pH gradient, and its consequent resolving power, can be altered by using ampholytes  covering  different pH  ranges.  The range pH 3-10 is used first to get an overview of where the proteins  focus and an appropriate choice of ampholytes can be made for a second round of focusing  over  a smaller range,  say pH  5  to  7.  The  smaller  the pH range covered, the greater will be the resolution.

2- Control of convection

Convective disturbances are currents induced in fluids by the effects  of gravity upon fluids which differ in density in different parts.  In  IEF  they are caused by heating effects, which reduces the  density of the heated solution, and from  the  fact that  focused protein  bands are more  dense than the surrounding ampholyte  solutions.  Practical  systems consequently require some way of obviating these  convective disturbances.

Since convection  depends upon  differences  in  density,  the  effect  of gravity and a consequent movement  of fluid, to avoid convection  any one or more of these three elements could be targeted.  Thus, an early approach was to conduct IEF in a sucrose gradient,  thereby  pre-imposing a density gradient which would damp out any  convective  disturbances.

To eliminate the effects of gravity,  IEF may be conducted in a rotating tube, so that the gravity vectors cancel out with time,  or,  at somewhat greater expense,  the  experiment  may  be conducted under microgravity conditions in outer space. Finally, because convective disturbances are a consequence of fluid movement,  another approach  is to  have  a system where the  liquid cannot  move,  such as in a gel, and a popular modern method is to conduct IEF in a gel slab.

3- Applying the sample and measuring the pH gradient

In any practical system, all  of the  attendant  problems must be solved simultaneously. Thus, in addition to controlling convection, the system must allow for sample application  - avoiding the  pH extremes  - and for measurement of the pH gradient  after  the  separation.  Different systems are suitable for analytical and preparative  purposes and one of each will be briefly described, by way of illustration.

3.1 An analytical IEF system

The most common analytical system in use at present  is the  thin  slab gel system.  In  this  a  thin  layer  of  gel - either large pore polyacrylamide or agarose  - containing an appropriate ampholyte mixture, is cast onto  a backing sheet of Gel-Bond ^Æ, a product  of FMC  Inc.  The  sheet  is positioned on top of a template on a cooling block.  The template  marks sample tracks,  and  serves  as a guide to  the  application  of the  sample

Wicks, made of several layers of filter paper, are impregnated with the appropriate acid or base electrode solution and placed on top of the gel at each end (Fig. 5).  When the  apparatus  is sealed, electrodes  contact  the filter paper wicks, thereby closing  the electrical  circuit (this description  is based on the Pharmacia Biotech Multiphor apparatus).

The pH gradient is allowed to become established before the  sample  is applied.  Sample is applied to the  gel by carefully laying small rectangles of filter  paper,  impregnated  with  sample  solution,  on  the  gel  over  a  track mark on the template.  If necessary, the  same sample solution can be applied at different positions, i.e. at different pH values, on the gel. Samples are drawn out of the  filter paper and into  the  gel by electrophoresis and by diffusion.  When this has happened the potential is switched  off and the  sample  applicator  papers  are  removed,  so  that they do not subsequently distort the electrical field.

Figure 5. Sketch of apparatus used for analytical flat bed IEF.

After focusing is complete, the gel is removed and stained to  reveal the position of the protein  bands.  Ampholytes  are polyamino compounds and at most  pH values they  will react with and precipitate dyes such as  Coomassie  blue.  To obviate this, the ampholytes can be removed by washing the gel in trichloracetic  acid before staining.  Asimpler method, however,  is to use the principle of the Bradford assay for protein, i.e. to stain with Coomassie blue G-250 in acid solution. Using this approach only the proteins are stained and the ampholytes do not interfere.

In order to  determine  the  pI values of the  separated proteins,  it  is necessary to measure the  pH gradient.  This can be done, after focusing but before  staining,  by using a surface-probing pH electrode.  However, as with other techniques, once  the  values for  a few proteins  have  been established, these can be used as  standards,  and the  values of unknowns can be determined by interpolation.  Standard proteins,  of known  pI values can thus be run in parallel with  unknowns -  on  the  same gel but in different tracks - and a standard curve of pH vs  distance  can  be constructed from the standards and used to determine the pI values of the unknowns. A table of pI values of standard proteins has been published by Chambers and  Rickwood, and pI calibration kits are commercially available.

3.2 Preparative IEF

Preparative IEF21 differs from analytical IEF in that it is done on a much larger scale, i.e. with much more sample and, as the products  sought are active protein fractions, provision must be made for recovery of the separated fractions after IEF.  The  central  problem  with preparative  IEF is stabilization  of the  system against convection  during the  focusing process while still being able to elute the separated components at the end of the  process,  Different  approaches  to  the  solution  of this  problem have been adopted by different authors and by the makers of commercially available apparatus.  The most common approach  is to conduct the  focusing  in  a convection-stabilized liquid phase.  With  the proteins and ampholytes being recovered in solution, measurement of the protein concentration and pH of each fraction  is relatively straightforward. An alternative approach, suited to smaller-scale preparative uses is to conduct the focusing in a slab gel, to cut out the focused band and electro-elute this. In this case the pH gradient would be measured as described for analytical IEF.

The first approach tried, and commercialized by Pharmacia, was to conduct the  focusing in a sucrose gradient,  which could be eluted  from  the apparatus at the end of the  experiment.  An ingenious, but expensive, apparatus had provisions for cooling the annular focusing column on both its inner and outer surfaces, and valves to  isolate the  electrode solutions during the elution phase.

An approach used by LKB (which has since merged with Pharmacia) was to use a flat bed of granulated gel.  After focusing, the gel bed could be divided into  a number of segments,  the  gel  from  each  segment  being scooped out and packed  into  a mini-column from which the  focused protein and ampholytes could be eluted.

In the  Bio-Rad Rotoforô  apparatus,  convection  is  controlled  by conducting the  focusing in  a horizontal  column  which is rolled at  1  rpm about its central axis.  Rolling serves to  negate  the  effects  of gravity  and enables the  proteins  to  be  focused  in  free  solution.  A  central  ceramic cold finger serves to  remove  heat.  The  column  is divided  into  20 segments by polyester  membranes.  After  focusing, solution  in the segments can be rapidly eluted from the side of the column into 20 test-tubes, without mixing.  The Rotofor is currently a favoured apparatus  for preparative IEF .

Although good results can be obtained by preparative  IEF,  and for many separations it may be the only practicable method, the technique  is constrained by the  high initial  cost of the  apparatus  and the  cost of the ampholytes consumed in each experiment.

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 .

Margolis, J.  and Kenrick, K.  G.  (1967) Polyacrylamide gel electrophoresis across a molecular sieve gradient. Nature (London) 214,  1334-1336.

Slate, G. G. (1968) Pore-limit electrophoresis on a gradient of polyacrylamide gel. Anal. Biochem. 24, 2 15-2 17.