about paper electrophoresis

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Presented by :- Pradnya Shinde.
M.Pharm –I year
Dept. P’ceutics
R.C.P. kasegaon.

2. The technique of paper electrophoresis is
simple and inexpensive and requires only
micro quantities of plasma for separation.
The support medium is a filter paper .
The electrophoresis apparatus in its simplest
form consists of two troughs to contain buffer
solution, through which electric current is
passed.
Frequently used in isolating proteins, amino
acids and oligopeptides.
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3. “The charge carried by a molecule depends on the pH
of the medium. Electrophoresis at low voltage is not
usually to separate low molecular weight
compounds because of dif fusion, but it is easier to
illustrate the relationship between charge and pH with
amino acids than with proteins (or) other
macromolecules.”
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4. The equipment required for electrophoresis consists basically of
two items, a POWER PACK and an ELECTROPHORETIC
CELL.
Power pack : provides a stabilized direct current and has
controls for both voltage & current output, which have an output
of 0 to 500V and 0 to 150mA are available.
The Electrophoretic cell: contains the electrodes,buffer
reservoirs, a support for paper and a supporting transparent
insulating cover.
The electrodes are usually made of platinum.
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5. The two arrangements of the filter strips are commonly
used. The horizontal & vertical arrangements. Both the
arrangements are equally viable & the choice usually depends
upon personal preferences.
Filter Paper:
Paper of good quality should contain at least 95% α- cellulose
and should have only a very slight adsorption capacity.
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6. 1)A long strip of filter paper is moistened with a
suitable buffer solution of the desired pH and the
sample is applied transversely across the central
part of the strip.
2)Ends are fixed to dip in buffer solutions in two
troughs fitted with electrodes.
3)Electric field of about 20 volts/cm is established.
4)The charged particles of sample migrate along
the strip towards respective electrodes of
opposite polarity, according to net charges, sizes
and interactions with the solid matrix.
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7. 5) Homogeneous group of particles migrate as a
separate band.
6) The electrophoresis is carried out for 16-18 hours.
7) Separated Proteins are fixed to a solid support
using a fixative such as Acetone or Methanol.
8) Proteins are stained (bromophenol blue) to make
them visible.
9) The separated proteins appear as distinct bands.
10) Drawback-long time interval and blurring of
margins.
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8. 1)The different fractions appear as blue
coloured bands across the filter paper
starting from the moving boundary
backwards.
2)If a quantitative estimation is required for
each fraction, the bands may be carefully
cut and eluted , or the bands may be
scanned optically in a densitometer.
3)In human plasma five different bands can
be identified on paper electrophoresis.
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9. Serum analysis for diagnostic purpose is
routinely carried about by paper
elelectrophoresis.
Muscle proteins, egg white proteins, milk
proteins & snake, insect venom analysis
done by this technique.
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