DEFINITION

Movement of charged particles (ions) in an electric field resulting in their migration

towards the oppositely charged electrode is called electrophoresis.

Principle :

It is widely used analytical technique for the separation of biological molecules such as plasma
proteins, lipoproteins.Charged molecules migrate either to cathode or anode depending upon the
kind of charge they carry. Molecules with a net positive charge (cations) move towards cathode
whereas molecules with net negative charge (anions) migrate towards anode.

TYPES OF ELECTROPHORESIS

Zone Electrophoresis:

a) Paper electrophoresis

b) Gel electrophoresis

c) Cellulose acetate electrophoresis

d) Thin layer electrophoresis

Moving boundary electrophoresis:

a) Capillary electrophoresis

b) Isoelectric focussing

c) Immuno electrophoresis
 REQUIREMENTS FOR ELECTROPHORESIS

Electrophoresis tank (to hold buffer).

Electrodes
Power pack to supply electricity at constant current & voltage. Power supply
vary based on type of electrophoresis and type of gel used. (PAGE usually uses
higher voltage while Agarose gel electrophoresis generally uses lower voltage)

Continued...

Buffer

To create pH mostly around 8.6. At this pH all

serum proteins will have net negative charge

& will migrate towards anode to conduct the current.

Specimen
(Serum, Plasma, Urine or Fluid CSF, Pleural fluid).
 FACTORS AFFECTING THE RATE OF MIGRATION

Total net charge on molecules.

-Size and shape of particles.

-pH of buffer solution.

-Strength of electric field applied.

-Property of supporting medium.

-Temperature

GEL ELECTROPHORESIS

-Gel electrophoresis is a method for separation and analysis of macromolecules like DNA,
RNA and proteins or their fragments, based on their size and charge.

-Gel electrophoresis uses a gel as an anti-convective medium and/or sieving medium during
electrophoresis.

-Gels suppress the thermal convection caused by application of the electric field, gels can
also simply serve to maintain the finished separation, so that a post electrophoresis stain
can be applied.
 PRINCIPLE OF SEPRATION

According to charge: When charged molecules are placed in an electric field, they migrate
toward either
the positive (anode) or negative (cathode) pole according to their charge.

According to size: The smaller molecules move more

swiftly than the larger sized ones, as the can travel through the pores more easily than the later.

INSTRUMENTS AND REAGENTS

-Electrophoresis apparatus

-Buffer

-Power supply

-Supporting media

-Detection and Quantification

 1-ELECTROPHORESIS APPARATUS

1-Electrophoresis chamber with buffer solution

2-Casting tray

3-Electrodes

2 . Buffer:
Buffers in gel electrophoresis are used to provide ions
that carry a current and to maintain the pH at a

relatively constant value.

The most common being, for nucleic acids Tris/Acetate/EDTA (TAE),

Tris/Borate/EDTA(TBE).

1- POWER SUPPLY

-The electrodes are connected to their respective terminals of the electrophoresis chamber and to the
power supplier with controls for rate of current flow.
-The best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm
to the gel
 SUPPORTING MEDIA (GEL)

-Starch

-Agar/agarose

-Cellulose acetate

-Polyacrylamide gel

-The kind of supporting matrix used depends on type of molecules to be separated and the
desired basis for separation: charge, molecular weight or both

Agarose and polyacrylamide gels are cross-linked,

spongelike structure
- It is important that the support media is electricall neutral. Presence of charge
group
may cause:

-Migration retardation

-The flow of water toward one or the other

electrode so called ‘Electroendosmosis (EEO)’, which decrease resolution of
the
separation.

AgaroseGelshave fairly large pore sizes and

are usedfor separating larger DNA molecules (RestrictionFragment Length
Polymorphism Analysis)

Polyacrylamide Gels are used to obtain

high resolutionseparations for smaller DNA molecules (STR analysisand
DNA sequence

)
analysis
 -Agarose and polyacrylamide gels are cross-linked,
spongelike structure
-It is important that the support media is electricall neutral. Presence of charge group may cause:

-Migration retardation

-The flow of water toward one or the other

electrode so called ‘Electroendosmosis (EEO)’, which decrease resolution of the separation.

AgaroseGelshave fairly large pore sizes and

are usedfor separating larger DNA molecules (RestrictionFragment Length Polymorphism Analysis)

Polyacrylamide Gels are used to obtain

high resolutionseparations for smaller DNA molecules (STR analysisand DNA sequence

)
analysis
 DETECTION AND QUNTIFICATION

-Stains

Protein staining

Ethidium bromide staining

-Blotting
Southern blotting (for DNA) Northern blotting (for RNA) Western blotting (for protein)
Process of Gel Electrophoresis
 VISUALIZATION

The molecules in the gel are stained to make them visible.DNA may be visualized using
ethidium bromide which, when intercalated into DNA, fluoresce under ultraviolet light,
while protein may be visualised using silver stain or Coomassie Brilliant Blue dye.
-SYBR Green I is more expensive, but 25 times more sensitiveand possible
safer than ethidium bromide.
-SYBR Safe is a variant of SYBR Green, and show low level ofmutagenicity
and toxicity.
Other less frequently used markers are Cresol red and Orange

APPLICATION

-Separation of Deoxyribonucleic acid

-Separation of ribonucleic acid

-Separation of protein molecules

-It may be used as preparative technique prior to use of other

methods such as mass spectroscopy, cloning, DNA

-Sequences, Southern Blotting for further characterization.

-Separation of amino acid

-Separation of lipoproteins

-Separation of enzyme in blood

-Separation of antibiotic drug

 AGAROSE GEL ELECTROPHORESIS OF SERUM PROTEINS
Aim: To perform electrophoretic separation of proteins in the given serum sample by Agarose gel
electrophoresis.

PRINCIPLE:

Electrophoresis is the movement of charged particles through an electrolyte subjected to an electric field.
The anions move towards the anode depending on the nature of their net charges. As a result of
differentrates of migration, a complex mixture such as plasma proteins can be separated into a number of
fractions; the sharpness of separation depends upon the extent of which each fraction is homogenous in its
mobility. Plasma proteins under experimental conditions of pH 8.6 exist as anions, thus they all move
towards anode.

REAGENTS

BarbitoneBuffer(pH8.6):1.84gofdiethylbarbiturateand10.3gsodiumdiethylbarbiturateisdissolvedinwaterand
madeupto1L.Or

Barbituricacidandsodiumacetatebuffer:Dissolve 5g sodium diethylbarbiturate in 34.2 ml of 100 mmol/LHCl

and 3.25g sodium acetate.Make up the volume to 1L with distilled water.

1. 1%Agarose:- 1g of Agarosein100ml of baritone buffer.Prepare fresh just before the use.Heat the
solution in a boiling water bath with occasional shaking until the viscosity is clear.
2. Fixative solution:– 99%Methanol
3. Stain:–0.5% Amido Black10B in 5% aceticacid.
4. Destainer:-5% Aceticacid.
PROCEDURE

PARTI:ELECTROPHORETICRUN

Preparation of Agarose gel slides:-

Place thoroughly cleaned,alcohol washed,glass microscope slides on a horizontal surface. Using a large fast
delivery 10ml pipette, layer about 1.5ml of the molten agarose onto the slide. Apply uniformly over the
slide, up to the edges on all sides so that the thickness of the gel is uniform over the entire slide. Allow
thegeltosetovertheslideatroomtemperaturefor3minutes.

Application of the sample:-

Cut thin filter paper (Whatmann no. 1 filter paper) strips (10X 0.3mm). Take one strip & moisten it with
test solution (serum). Excess sample is removed by pressing in between the filter paper. If two samples are
to be applied on a double size slide, the sample strips should be applied at a distance of 1cm from each
other without any time lapse. Place the strip in the center of the prepared Agarose gel slide 1.5 to 2 inches
from one edge (cathode end) in order to prevent contact of sample with buffer. Sample strips on the gel
should be pressed gently with forceps. Immediately after the application of the sample strips, the slide
should be placed in the buffer tank.

Resolution of serum proteins

Switch on the electrophoresis power pack 15minutes before the start of the run. In the electrophoretic
tank, put chilled buffer in both the compartments to the required level. Place frozen coolant sponges in
the inner chamber (slide area) of the tank. Place wicks of Whatmann paper of same width as the slide in
the tank to provide connectivity of the buffer with the gel from both the sides. The gel slide and the buffer
wicks
areplacedhorizontallysuchthatthesideoftheslidetowhichserumisappliedistowardsthecathode.Closethe
chamber and connect the terminals to the power supply. Adjust the voltage to 150V (so that a current of
7mA per slide is obtained). The voltage should be maintained throughout and fluctuations avoided. Keep
frozen gel ice packs on top of the chamber to minimize heating effects on the gel. Run electrophoresis for
90 minutes, then switch off the current and remove the slide from the tank.

PARTII: PROCESSINGTHEPATTERNONTHESLIDE

Fixation of proteins in agar gel over the slide:

Soak the slide in the fixative (methanol).The native proteins separated over the gel will be fixed in their
areas of separation. Remove the slide fixed in their areas of separation. Remove the slides after 10-
15minutes.(The gel over the slides appear completely opaque now)
Dehydration of the gel:-

Place the slide in a hot air oven at 70oC for 20-30 minutes.(the slide becomes totally transparent). During
dehydration most of the buffer salts will be removed and the agarose gel is fully dried and fixed to the slide.
Too much of over heating is avoided to avoid the peeling off of the gel.

Staining:-

Remove the slide from the oven and cool to room temperature. Then soak the slide in the stain (0.5%
amidoblack) for15 minutes in order to stain protein fractions.

De-staining:-

Wash the slides by immersing them three times in fresh portions of 5% acetic acid for 5 minutes each. Now
the protein fractions over the slide are clearly visible. Rinse for 5 minutes in water and dry in a hot air
ovenat70oCfor5minutes.

Note:

The buffer can be used (for20to30 times) until it becomes turbid.

These run sample should be uniformly absorbed by the strips (Whatmannno.1filterpaper).

The gel prepared can be stored in the refrigerator and can be used by boiling in the water bath for 5minutes.

Result:

The electrophoretogram obtained shows pattern.Albumin,α1,α2,β and γGlobulins

Interpretation:

In anormal electrophoretogram, the proportions of various bands are

Normal band % of total protein Conc.g/dl

Albumin 50-70% 3.5-5.2g
α1globulin 2-6% 0.1-0.4g
α2globulin 5-11% 0.4-0.8g
βGlobulin 7-16% 0.5-1.2g
γglobulin 11-22% 0.7-1.5g
The main component of α1 region are α1 antitrypsin, α1 acidglobulin, α1 lipoprotein, α1 fetoprotein.

The main component of α2 region are α2macroglobulin, haptoglobulin,etc. In some disease,ceruloplasmin

also makes a significant contribution.

The β band (β1&β2)consists mainly of transferring and β2 globulin.Italso

hashemopexin,complement4,complement3,β lipoprotein and fibrinogen.

If plasma is used fibrinogen forms an intense band between the β and γ

bands. γ band consists of immunoglobulin and C-reactive protein.

Clinical Significance:

The electrophoresis of serum proteins is helpful in detecting the changes in individual protein fractions in
serum and in detecting abnormal bands in certain diseased conditions.

In chronic infections, the γ globulins are increased but the increase is smooth and wide based. In para
proteinemia a sharp spike is noted and is termed the M- Band. This is due to the monoclonal origin of
immunoglobulin in multiple myeloma. In primary immune deficiency, γ globulin fraction is reduced. In
nephritic syndrome, all proteins except very big molecules are lost in urine so α2 fraction (containing
macroglobulin) will be prominent. In cirrhosis of liver albumin synthesis by liver is decreased with narrow
albumin band and polyclonal elevation of gamma globulin band merging with β globulin band is
seen.Inα1antitrypsindeficiencyα1bandisdecreasedorsometimesmissing.
Disease Albumin Globulins
1 2  
Nephroticsyndrome    - 
Cirrhosis ofliver  - - - 
Kwashiorkor  - - - 
Hypogamma Globulinemia - - - - 
A gamma Globulinemia -  - - Absent
Multiplemyeloma - - - - 
Kala-Azar - - - - 
Diabetes mellitus - -   -
Viral hepatitis  - -  

Normal and abnormal Electrophoretograms