Electrophoresis.

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ELECTROPHORESIS

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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Electrophoresis
Electrophoresis may be defined as separation of
charged compounds using electric field.
Rate of movement of compounds depends upon charge
and size of the compound
Serum protein electrophoresis pH 8.6
Proteins negatively charged (an ions) move
towards anode
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Applications
1. Separation of serum proteins
2. Separation of serum lipoproteins Hyper / Hypo
lipoproteinemias
3. Separation of Hemoglobin variants
Hemoglobinopathies
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4. Separation iso Enzymes - LDH, CK
Classification of Electrophoresis
1. Moving boundary electrophoresis.
Described by TISELIUS. He used a U shaped tube
and using a buffer separated plasma proteins
into 4 fractions, Alb, a, ß, ? . Not used now
because of poor resolution.
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Zone Electrophoresis
This is the most commonly used type of
electrophoresis. Sample is applied as a band or
spot on chemically inert and homogenous support
medium on which separation occurs as zones /
bands. Based on support medium used zone
electrophoresis is sub classified as
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1. Paper electrophoresis
Cheapest method but the disadvantage is some
mixing occurs between different zones. Requires
several hours. Separation is not very sharp.
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2. Cellulose acetate membrane electrophoresis
There is clear separation into discrete zones.
Requires about one hour time.
3. Gel electrophoresis
A) Agar gel is commonly used for immuno
electrophoresis.
B) Starch gel is commonly used for separation of
iso enzymes
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C) Poly acrylamide gel electrophoresis
(PAGE). Maximum number of protein bands are
obtainted in this technique.
Factor affecting separation
1. Charge of the compound
2. Size of the compound
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Electrophoresis Unit
Electrophoresis chamber consists of two
compartments separated from each other by a
dividing wall, one side contains the anode and
the other the cathode.
Each side is filled to the same level with a
buffer ( barbital buffer, pH 8.6)
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A bridge across the top of the dividing wall
holds a cellulose membrane or other support
material like filter paper, agar gel etc., so
that each end of it is in contact with the buffer
in one of the compartments.
The only connection between the two compartments
is by the way of the membrane.
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First membrane is immersed in buffer and placed
in the chamber and the sample is applied.
When a voltage is applied to the cell the current
is carried across the porous membrane by the
buffer ions.
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At. pH 8.6 all the serum proteins carry a net
negative charge and tend to migrate toward the
anode.
Albumin carries the largest charge and therefore
moves the fastest.
The ? globulins have the smallest net charge
and move the least distance.
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Densitometric Scanning
After separation of the compounds by
electrophoresis and staining the compounds
whether the pattern is normal or abnormal can be
visualized. For more accurate fractination
densitometry is very valuable
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After completion of electrophoresis, the
supporting medium is placed in a fixative ( 7
acetic acid), to prevent diffusion of separated
fractions.
Separated fractions are then visualized by using
appropriate stains, e.g.
Bromophenol blue and amido schwartz for plasma
proteins and
Sudan black for lipoproteins.
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Quantitation is done by
Densitometry or
Elution, followed by colorimetry or spectro
photometry of the eluted fractions.
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Serum protein electrophoresis
Albumin 55 65 Globulins 35 45
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a 1 globulins 2 4
a 1 Acid glycoprotein ( oromucoid) 0.6 1.4
gm/dl Carbohydrate content is 41. Oromucoid is
considered as an acute phase reactive protein.
Increased in acute inflammations cirrhosis of
liver. It binds to progesterone and transports
it. Carries carbohydrate to the site of injury
for repair.
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a 1 anti trypsin ( a1 AT) 200 400 mg / dl.
Active elastase a1 AT
inactive
elastase Decreased in emphysema, cirrhosis of
liver
a 1 Lipoprotein (HDL)
AFP ( if present) principal foetal protein normal
plasma concentration less than 1 micro gram per
dl. Increased in hepatocellular cancer.
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a 2 globulins 6 12
Haptoglobin - Synthesized in liver. Binds free
hemoglobin and prevents its loss through kidney /
urine. Heptoglobin concentration is decreased in
hemolysis.
Caeruloplasmin contains copper blue coloured
protein. Molecular weight 151000. It has 8 sites
for copper binding. Normal concentration 30 mg /
dl. Decreased in Wilson's disease.
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ß globulins 8 12
Transferrin - Transports iron in plasma.
Increased in iron deficiency anemia.
Hemo pexin Binds with heme and prevents its
loss through urine
ß Lipoprotein (LDL)
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? globulins 12 22
Changes in serum protein pattern
The Acute Phase Reaction
Acute inflammation / tissue necrosis there is
increase in serum levels of a1 acid
glycoprotein, a1 AT, ceruloplasmin. Fibrinogen, C
reactive protein and haptoglobin. Increased ESR
is due to increased fibrinogen and other
globulins.
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Acute Phase Reaction
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Para Proteins (M Proteins)
M refers to Myeloma / Monoclonal globulins. para
protein may be whole or part of immunoglobulin.
If the para protein is of light chain it may
spill in to urine (Bence Jones proteinuria)
Bence jones proteins precipitate around 40
600 C and then redissolve at higher temp. These
proteins appear as sharp peak ( spike ) mainly in
? region
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Multiple Myeloma
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Causes Multiple myeloma (Plasma cell myeloma),
lymphoma paraprotemias are associated with
decreased ? globulins and albumin.
Cirrhosis of Liver Albumin Decreased, ?
globulins increased.
ß ? bridging due to IgA increase
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(No Transcript)
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Separation of lipoproteins
Chylo - Microns
ß
Preß
LDL
a
VLDL
HDL
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Separation of Iso enzymes of LDH

MI LDH 1 LDH 2
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