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ELECTROPHORESIS

Introduction
A separation technique of different
charged
molecules in solution in an electrical field of
varying Potential.

Used in the clinical laboratory for the


separation of:-
(1)Proteins.
(2)Isoenzymes (e.g. creatine kinase, LDH ,
etc.)
(3)Haemoglobin
General principle
Electrophoresis is based on the
movement of charged molecules in solution
in an electrical field.

Depending on the nature of the net


charge, the charged particles will migrate
either to the cathode or to the anode.
The molecule will move in the direction of
its opposite charge.

Negatively charged molecules (anions)


will move toward the positively charged
electrode, (the anode).

Likewise, positively charged molecules


(cations) will move toward the (cathode),
the negatively charged electrode.
Cathode Anode
Separation of charged particles during
electrophoresis occurs when different
molecules move at different rates.

Factors influencing the rate of migration:-


(1)Support media.
(2)Buffer.
(3)Voltage , current and heating effect.
(4)Charges , Size and shape of particles.
(1)Support media
The support medium provides the matrix
in which separation of molecules takes
place.

Various support media are used e.g.


Starch , Agrose , Polyacrylamide , Cellulose
acetate.

Some effects seen with :-


(I)Adsorption can lead to tailing effect.
(II)Electro-osmosis (with starch, agar and
(2)Buffer
Have two purposes in electrophoresis:-
(I)Carry the electric current.
(II)Fixing the pH and determine the charge
on the molecules.

Different buffers are used depending


upon the conditions of experiment.

For the separation of serum proteins


barbital or tris borate-EDTA buffers remain
most popular.
Buffer composition affects migration rate
by interacting with proteins.

The rate of migration increased with


decrease in ionic strength of the buffer but
the band become more diffuse at low ionic
strengths , probably due to impaired
buffering capacity.
(3)Voltage , current and heating
effect
Themigration rateis directlyproportional
to voltage , current.

The increase of voltage leads to


increases in heat.
(4)Charge , Size and shape of
particles
Charge :-migration rate increases with
increase charges of molecules.

A larger and more asymmetric molecule


will have a slower rate of movement in the
electrical field due to friction.

Shape:-Fibrous or globular proteins


exhibit different migration rates.
Components
Power supply
Designed to provide a constant voltage.

Some power supplies also have the option


of providing constant current or constant
power.

Electrophoreses are typically performed


bwteen 50 & 200V.

Higher voltage improver resolution and


decrease the time needed for separation.

Voltage cant be increased to much ,


Serum Protein
Electrophoresis
Should be performed on serum from
clotted blood , because the presence of
fibrinogen may mask , or be interpreted
as , an abnormal band or protein.

Method
(1)Mark the acetate strips in pencil at the
point of application of sample application of
sample about 4 cm from the cathode end.
(2)Add some TEB buffer to a shallow dish
and carefully float the strip on top so that it
is impregnated with buffer from below by
capillary action.

(3)Rmove excess buffer from the strip by


blotting lightly on filter paper do not
overdry by pressing too hard.

(4)Take 0.5ml of sample using micropipette


or by capillary tube and placed in strip
(5)Place the strip between the two shoulder
pads of the electrophoresis tank, with the
origin in the
centre and the end with your name towards
the anode and carefully pull taut.

(6)Electrophoresis will be carried out at a


constant voltage of 150 V (approx. 0.5
mA/cm strip width) for 60 mins.
Detection & Quantification
After separation the molecules are
detected either by:-
(1)Staining followed by quantification using
a densitometer.
(2)By direct measurement using an optical
detection system.

Normal pattern
Multiple myeloma
Urine
Electrophoresis
The urine sample needs to be
concentrated due to the fact that there is
usually a small amount of protein in urine.

There are two methods for concentrating


urine:-
(I)Lyphogel method.
(II)Mincion method.
(I)Lyphogel method
Lyphogel is a polyacrylamde hydrogel
which absorbs water and low molecular
weight substances from biological fluids.

Add 1.48g lyphogel to 10ml of urine in a


test tube.

Cover the test tube and leave for at least


5 hours.

Remove the gel pellet from the solution


using forceps.
(II)Minicon method
This method uses a chamber lined with
an ultrafilitration membrane .

The membrane allows passage of solids


up to a molecular weight of about 15000.

Place 5ml of clear urine through the hole


in the top of the chamber using a Pasteur
pipette.

Within 4 to 5 hours , the urine will be


Lipoprotein
Electrophoresis
Electrophoresis separates lipoprotiens on
the basis of their charge and mass to yield
four major bands.

Lipoprotein in serum can be separated by


electrophoresis into four different bands.

They are named according to their


electrophoretic mobility as , pre and
which correspond to HDL , VLDL and LDL
respectively.

Chylomicrons remain at the point of


application.
Haemoglobin
Electrophoresis
(A)Blood collected into EDTA.
(B)Centrifuge & remove the plasma.
(C)Preparation of haemolysate (Lysate):-
(1)Wash the cells three times using normal
saline.
(2)Take two volume of & one volume of
D.w.
(3)Wash using carbon tetra chloride (CCl4)
to extract cells.
(4)Centrifuge prepared lysate.
Maintenance and QC
Elecrophoretic methods require rigid
adherence to a well-documented procedure
so that reliable , reproducible results will be
obtained.

Proper maintenance involves:-


(1)cleaning of electrodes and buffer
chambers B/W uses.
(2)Replenishment of staining solution.
(3)Performance of linearity , wavelength
and photometric accuracy and stray light
checks on the densitometer.