Paper electrophoresis

One of the  earliest forms of zone electrophoresis  for the  separation of proteins  was  paper  electrophoresis.  In this a strip of filter paper was used as a medium to support a thin layer of buffer.  Since the paper served only to support the  buffer, paper electrophoresis  can be considered as a form of free electrophoresis (as opposed to electrophoresis in a sieving gel)  The experimental  set-up for paper electrophoresis is shown in Fig. 1.

A strip of filter paper, typically 20 x  150 mm,  is marked with pencil to indicate the  anodic and cathodic ends and a line is lightly drawn transversely in the middle, where the sample is to be applied.  The strip is soaked in buffer, blotted  briefly  and suspended between  supports  in the apparatus. Buffer is added to both the anode and the cathode compartments:  it is important that the levels in the two compartments are the  same to  prevent  siphoning through the  filter  paper.  The  filter paper is connected to the buffer by  filter  paper wicks, which must be the same width as the filter paper  strip,  but can  be made of several  layers of filter paper.

Figure 1. Diagrammatic cross-section of an apparatus for paper electrophoresis.

Sample can be applied as a thin  line  across the  middle of the  paper strip, but not within ca. 5 mm of the  edges.  There  are  different  ways of applying the  sample: a simple way, but which requires some  manual dexterity, is to use a Pasteur pipette, drawn down to a thin  capillary. After application of the  sample, the  apparatus  is sealed with a lid  This enables the  atmosphere  within  the  apparatus  to  become  saturated  with water vapour,  thereby preventing  evaporation  of water  from  the  buffer on the  strip.  The  buffers have a maximal exposed surface  area to encourage rapid equilibration of the water vapour.  As a safety precaution. the apparatus lid is usually coupled with the  electrode connections, so that removal of the lid breaks the electric circuit. Without this precaution, fatal shocks might result from  inadvertent contact with the electrode  solutions.  Electrophoresis  is  run,  usually for  a number of hours,  typically  using  a  voltage  gradient  of ca. 10 volts cm^-1.  To keep electrolysis products away from the protein  samples being separated. the  buffers in the  electrode  compartments  are separated from the wicks by a baffle system.

After separation, the protein bands are fixed in position  and stained with a protein-specific stain, such as Ponceau S or Amido Black, and destained. Paper electrophoresis was used in medical diagnostics and a typical result for the separation of serum is shown in Fig. 2.

Figure 2. A typical separation of human serum by paper electrophoresis at pH 7.2.

Electroendosmosis

Paper is comprised largely of cellulose, a β →1 linked polymer of glucose. Glucose is hydrophilic due to the polarity of its many  -OH groups. Which readily form  hydrogen  bonds with water.  Cellulose as  a whole is not water soluble, however, because of its  extensive  interchain hydrogen bonds.  In forming hydrogen bonds with water, the H of the - OH groups of glucose is shared with the oxygen of water, giving the water a δ+ charge and the cellulose oxygen a δ - charge.

Cellulose thus  acquires an  overall  negative  charge,  called  the  “zeta potential”. When  placed in an electrical field, a strip  of paper  (cellulose) tends to move towards the anode, but cannot do so  as it  is fixed in  place.

The hydroxonium ions, H³O⁺, however,  are free to  move towards the cathode and do so, resulting in a net drift of the buffer towards the cathode. This drift of the buffer towards the cathode, known as Electroendosmosis, increases the apparent mobility of cations and decreases that  of anions.  For example,  in Fig. 2, γ globulin is  seen  to have an apparent migration to the cathode.  However, the pI of γ globulin         is 6.8,  and so at  pH  7.2  it  would be  expected  to  have  a net negative charge and a consequent anodic migration.  In fact it does have a slight anodic migration but the electroendosmotic flow is faster than  this, resulting in the apparent cathodic migration.

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 .