PAPER ELECTROPHORESIS

In paper electrophoresis, the supporting used is a chromatographic paper which is cut to

the required size to carry out electrophoresis. Low voltage paper electrophoresis is simple, cheap
and used for separation amino acids, peptides, proteins, nucleotides, nucleic acids & charged
carbohydrates.

Equipment: Horizontal electrophoresis equipment.

Operational procedure:-

Disadvantages:

1. Adsorption:
But adsorption is minimized by using buffer, more alkaline then isoelectric point
of sample.
2. Endo – osmosis:
3. Diffusion of small molecules:
It is overcome by applying high voltage (~ 20 V cm-1) thereby reducing the time
required for separation. In case of high voltage paper electrophoresis, the paper is
clamped between 2 cooled plates to carry off the heat generated by the high voltage
current flow.

CELLULOSE ACETATE ELECTROPHORESIS

High purity cellulose acetate is commercially available in thin uniform strips

which have a uniform micro pore structure.

Equipment: Horizontal electrophoresis equipment.

Operational procedure:-

Advantages:-

1. Very little adsorption even with macromolecules. Therefore suitable supporting medium
for separation of radiolabelled substances, immuno-diffusion & immuno-electrophoresis.
2. Better resolution in shorter time compared to paper electrophoresis has less hydrophilic
than paper & less buffer is held by it.
3. Thin ground of strips may be rendered translucent (permitting light to pass through
partially) after separation & staining by treatment with clearing agents such as whitemor-
oil 120 which facilitates quantification.

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 1

Disadvantages:-

1. Not used for preparative work.

2. Since low buffer is held by the strips, care must be taken to prevent.
3. Trips from drying.

Applications:-

1. Separation of compounds like amino acids, peptides, nucleotides, nucleic acids &
charged carbohydrates.
2. Particularly used for separation of blood proteins like glycoprotein, lipoprotein &
hemoglobin.

STARCH GEL ELECTROPHORESIS

Gels are water insoluble, hydrophilic & semisolid colloids prepared shortly before use.
Molecular sieving property of semi–rigid gel helps to separate large ionic compounds such as
proteins which have similar charge properties but differ in size & shape.

Starch gel is prepared by heating & cooling partially hydrolyzed starch in an appropriate
buffer. This causes the branched chain of amylopectin component of starch to intervene & form
is semi–rigid gel.

Weak high porosity gel is prepared by less than 2% (w/v) starch.

Strong less porosity gel is prepared by less than 8–15% (w/v) starch.

Equipment: Horizontal/vertical electrophoresis is used.

Operational procedure:-

Applications:-

1. Separation of complex mixtures of structural molecules & physiologically active

proteins.
2. Analysis of isoenzyme pattern.

AGAR GEL ELECTROPHORESIS

Gels are water insoluble, hydrophilic & semi–solid colloids prepared shortly before use.
Molecular sieving property of semi–rigid gel helps to separate large ionic compounds such as
proteins which have similar charge properties but differ in size & shape.

Agar is a cheap, nontoxic & chemically well defined. The powdery mixture of agrose
containing two galactose based polymer – agrose & agropectin. Agar is soluble on boiling in
aqueous buffer to perform a gel of about 380C.

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 2

Equipment:

Operational procedure:-

Advantages:-

1. 1% (w/v) high water content,

 good fibre structure,
 large pore size or
 Frictional resistance.
Therefore movement of ions is rapid during electrophoresis &
macromolecules can be separated.
2. Low diffusion make it excellent for separation and detection of antigenic proteins by
isotachophoresis & immunoelectrophoresis.
3. Purified agrose gel is used for separation of nucleic acid and high molecular weight
proteins because of it lack of molecular sieving & elctro-osmosis.

Disadvantage:-

Electro-osmosis is severe due to presence of sulphate group.

Applications:-

Analysis of

 Serum protein,
 Hb varients &
 Isoenzyme pattern.

Operational procedure (for paper, cellulose acetate, agar & starch):-

1. Medium saturation:

If the supporting medium is gel, the medium of saturation is a necessary because, the
gel is saturated with buffer at the time of gel preparation.

If the supporting medium is not a gel, it is saturated with buffer before

electrophoresis, since buffer conducts majority of current.

If paper is used as a supporting medium, it is saturated with buffer as follows: the

paper is dipped in buffer and excess buffer is removed by blotting.

If cellulose acetate is used as supporting medium. it is saturatted with buffer as follows.

Cellulose acetate strips are with buffer by floaing them on the surface of shallow tank.
(Saturated)

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 3

Sample Application:

Sample is applied by using micropipette as a small spot or narrow greak on or along the
suitable origin.

For paper and cellulose acetate: If the individual components of a miximum have
opposite charges. They migrate in both directions. The before sample is near the middle of the
supporting medium.

If the components are either positive / negative charged samples,they are applied near
end, farthest away from electrode to which they are moving.

For gel: In gel electrophoresis, sample is introduced to get stabs usually (or) a strip of
filter paper into narrow stabs cut into the gel surface.

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 4

Sample Volume: In microelectrophoresis, 0.1 ml - 1 ml of sample is used and in preparative
electrophoresis, more than 5 ml of sample is used.

Sample concentration: In electrophoresis, the sample concentration will be within the range of
1 - 3 mg / ml.

Urea / SDS are added to protein to facilitate their solubilisation.

In gel electrophoresis, bromophenol blue is used as tracker dye. By observing the

migration of the dye, gives some indication about the behavior of the system.

Running of the Sample:

After applying the sample, power switched on (at required Voltage) for a period
necessary for separation.

Voltage for low voltage elecctrophoresis 20 v.cm-1

Voltage for high voltage elecctrophoresis 200v.cm-1

Careful watching of equipment is necessary because overheating and chearing of paper

may occur if the supporting medium is not observed properly. If heat is expected, the entire
apparatus is kept in cold room. (0-4oC)

Time required for--

High voltage separation - Below / hour

Low voltage separation – 2 hours

The electrophoresis is completed the power is off. And the supporting medium is
removed.

Removal of buffering medium:

Paper cellulose acetate strips removed and dried and kept oven at 110.C. (unless
hermoliable compounds are present)

Gel stabs removed without any pressure. Cylindrical is removed by forcing water from
hypothermic syringe around the walls supporting glass tubes allowing the gets to be excluded
under gel pressure

After removal, gel is immersed in 7% acetic acid (which is used as fixative) to minimize
diffusion of compounds.

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 5

Detection, Recovery and estimation:

Unknown compounds identified by comparing the migration characteristic of standard.

If sample absorb or fluoresce under uv and support medium not agents they can be
identified.

If separation compound shows uv absorption after treatment with agents they can be
identified.

Eg: Amino acid, Peptide, Protein, fluoresce on reacting with dansyl cholor

If on treatment with reagents if the biological molecules produce structural coloured compound
they can be analyzed.

Enzyme Detection:

Method:1

Detected by immobilizing the non - ionic substrates during gel preparation and the
enzyme activity detected after electrophoresis incubation in a more appropriate buffer.

STEP: 1 STEP: 2

STEP: 3 Electro cardio gram incubated in appropriate buffer.

Method: 2

Enzyme separated (electrophoresis) is kept in close cont. with the supporting medium
containing substrate. The enzyme activity is localised.

If the separated compound is radioactive, autoradiography or radiochromogram scanner

may be used.

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 6

METHODS FOR ELECTROPHORESIS:

HORIZONTAL TYPE:

Electrophoresis equipment required for horizontal system basically consist of two items

*Power pack

* An electrophoresis

POWER PACK:

The power pack provides a stabilized direct current and it controls both voltage (0 -
500V) and current output (0 - 150 A)

Electrophoresis Unit:

The electrophoresis unit consists of

-- Electrodes
--Buffer reservoir
--Supporting medium
--Transparent insulating cover

Electrodes are made up of platinum or stainless steel

There are two buffer reservoirs and each buffer reservoir consist of two compartments
separate compartments are necessary, because any changes in pH occurring at electrodes does
not affect the buffer that is in contact with the supporting medium.

Support for electrophoretic medium (Supporting medium) may be paper, cellulose acetate
or gel. The contact between the supporting medium and buffer reservoir through 'wick'.

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 7

 Transparent insulating cover provides electrical insulation and also prevents evaporation
of buffer.

The supporting medium is cast on a glass / plastic sheet and placed on a cooling plate. (
an insulated surface through which cooling water is passed to conduct away generated heat.

VERTICAL TYPE:

Electrophoretic equipment required for vertical system basically consist of two items.
A power pack and
An elactrophoresis unit

POWER PACK:

The power pack provides a stabilized direct current and it controls both voltage (0-500V)
and current output.

ELECTROPHORESIS UNIT:

The electrophoresis unit consists of

--- Electrodes
--- Buffer reservoir
--- Supporting medium
--- Transparent insulating chamber
Electrodes are made up of platinum or stainless

There are two buffer reservoirs; upper buffer reservoir and lower buffer reservoir.

The supporting medium is gel especially poly acryl amide gel. The gel is formed between
two glass plates that are clamped together but held apart by plastic spacers. The plastic comb
(well forming template) is placed in the gel solution and is removed after polymerization to
provide loading wells for samples.

Gel dimensions are typically 12*14 cm with a thickness of 1 - 2mm.

Transparent insulating chamber provides an electrical insulation.
When the apparatus is assembled, the electrophoresis tank buffer surrounds the gel plates
and affords some cooling of the gel plates.
During electrophoresis the upper part of the gel is in contact with upper buffer reservoir
and lower part of the gel contact with the lower buffer reservoir.

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 8