PAPER ELECTROPHORESIS
Introduction
Electrophoresis is another separation technique. It is based on the different
mobility of ions (molecules which are charged) in a support which is
subjected to an electric field. Ions run more or less quickly along the
substrate according their charge, size, shape, etc. According to the
technique which is used, the apparatus consists of two small basins which
contain an electrolyte, a support (i.e.: filter paper, cellulose acetate strips,
polyacrylamide gel, or a capillary tube), an electrical DC power supply and
two electrodes. Electrophoresis is widely used to separate substances such
as amino acids, proteins, strands of ADN, etc. As in the case of
chromatography, people use different supports and solvents according to the
substances to be separated and the techniques used. A useful reference is
found in the magazine "Scientific American" about paper electrophoresis (4).
Method
As shown by the figure 21, inspired by this article, place two little basins a
few cm (one inch) apart. Pour in the basins an electrolyte made of a
teaspoon of table salt and another of baking soda in 300 ml (1 1/2 cups) of
tap water. Place a glass plate on the two basins and on it place a stripe of
filter paper soaked with the electrolyte. This stripe has to be immersed with
each end in the electrolyte in the basin for to complete the electrical circuit.
With a pencil, draw a line across the filter paper and place a little drop of
blood on it. Cover the paper with a second glass plate. Put an electrode into
the electrolyte of each basin and apply 45V in direct current (from 4 to 8
Volt per cm). You can obtain this from 5, 9volt batteries connected in series.
With the passing of time, you should see 5 little spots move toward the
negative electrode. These spots are made of different proteic components of
the plasma: globulins (alpha, beta, gamma), albumin and fibrinogen. In
reality, to make these substances better visible it is necessary use a stain
such as bromphenol blue. Lacking it, try red cabbage juice.
2 - Try also to separate the components of the substances already used in
the chromatography experiments and observe what it happens. Keep in
mind that some of these substances may not be ionic.
Caution!: Do not use high voltages for this experiment. Never touch the
electrodes, the electrolytes, or the paper stripe. Take care never to short out
the two electrodes. Finally, we recommend to insert a fuse on one of the two
cables. The fuse will break the circuit when there is too high a current. We
suggest about 10 mA. At the end of the migration of the spots, remove the
electrodes from the basins and take off the cables from the batteries.
References
http://www.chemistry.adelaide.edu.au/external/SocRel/Content/electrop.htm Electrophoresis (an introduction)
http://www.chemistry.adelaide.edu.au/external/Soc-Rel/Content/capel.htm Capillary Electrophoresis (CE)
http://a32.lehman.cuny.edu/molbio_course/agarose.htm Agarose gel
electrophoresis for ADN
http://a32.lehman.cuny.edu/molbio_course/overview.htm Molecular
Biology Course Overview
http://faculty.uca.edu/marc.hirrel/bio1/GelElectro2.htm
Electrophoresis
Internet keywords: electrophoresis introduction.
Bibliography: 1 p184. 1 p 207. 4.
Protein Gel