It’s a technique based on the movement of ions in an electric field.
It is used to separate the proteins in serum, urine, and other body fluids (mainly cerebrospinal fluid)
Positive charged ions moves towards the cathod (negative electrode).
Negative charged ions moves towarda the anode (positive electrode).
Migration of ions have different rates & thus separated.
3. ELECTROPHORESIS
• It’s a technique based on the movement of ions in an electric field.
• It is used to separate the proteins in serum, urine, and other body
fluids (mainly cerebrospinal fluid)
• Positive charged ions moves towards the cathod (negative electrode).
• Negative charged ions moves towarda the anode (positive electrode).
• Migration of ions have different rates & thus separated.
• Mobility of a particle is directly proportional(related so that one becomes larger or
smaller when the other becomes larger or smaller) to voltage applied & net charge of
particle.
• Mobility of particle is inversely proportional to the friction offered by
the particle in an electric field depending upon molecular size &
shape.
5. Mobility => directly proportional=> voltage applied+ net charge of
particle
Mobiloity => inversely proportional => Friction by the particle in electric
field (dependent on size+shape)
6. o INSTRUMENTATIONS
It is consists of a the
following parts:
i. High-voltage supply,
ii. Electrodes
iii. Buffer.
iv. Support for the buffer
(e.g filter paper).
v. Cellulose acetate strips.
vi. polyacrylamide gel or a
capillary tube.
M E D I A F O R
E L E C T R O P H O R E S I S
i. Paper (obsolete)
ii. Cellulose acetate
membrane (CAM).
iii.Gels
a. Starch Gel
b. Polyacrylamide Gel
(PAGE)
c. Agar Gel
d. Agarose Gel
7. o CELLULOSE ACETATE MEMBRANE (CAM).
• An electrophoresis chamber or tank consists of two compartments separated by a partition.
• Each compartment has an electrode made of an inert material such as platinum.
• Each side is filled with equal amount of a suitable buffer solution.
• A bridge across the top of the partition holds a membrane or gel with each end of it in
contact with the buffer directly or through paper wicks()ٹکڑے.
• The only connection between the two compartments is through this membrane.
• Sample is applied on to the membrane. Electrical power source is attached to the tank,
which has an indicator for polarity(the relative orientation of poles; the direction of a magnetic or electric field).
• Current of prescribed voltage is applied.
• Molecules start migrating through the membrane to anode or cathode depending upon their
net charge.
• After the prescribed time, current is switched off and the membrane or gel is removed from
the tank.
• It is then treated with suitable fixative and is stained to make the separated bands visible.
8. o REAGENTS:
• Cellulose acetate strips.
• Barbitone buffer (pH 8.6, ionic strength 0.05.Dissolve 10.16g sodium barbitone
and 1.84g diethylbarbituric acid in about 800 ml water and make up to 1L).
• Fixative solution {prepared by dissolving 5g trichloracetic acid (TCA), 5 g zinc
sulphate (ZnSO4) and 0.35 g sulphosalicylic acid (SSA) per 100 ml distilled
water}.
• Ponceau(ponsau) S, 0.5% w/v in 5% trichloracetic acid. (Other protein stains
such as commassie brilliant blue (CBB) or amido black can also be used.)
• Destaining solution (Acetic acid, 5% v/v in water).
• Clearing solution (prepared by adding 15ml glacial acetic acid in 85 ml
methanol. This solution is corrosive and volatile, therefore minimum amount
needed should be prepared with precautions.)
9. o PROCEDURE:
• The cellulose acetate strips are marked with lead pencil and
soaked in running buffer in a shallow tray avoiding inclusion of
air bubbles under the surface.
• Soaked strips are lightly blotted( پھوال
ہوا ) to remove excess
buffer.
• The strips are placed over the bridge or supports in the tank
and wicks of filter paper are placed over both ends to dip into
the buffer.
• From 3-5 μl sample is applied near the cathode in a row
leaving spaces in between and clear margin on either side.
Replace lid and connect power supply.
10. • The current is adjusted to 0.4 mA per cm width of strip
(~185V). Run for 20-60 min. Time and voltage or current
varies with different apparatuses and procedures. After
completion of electrophoresis, the strip is removed, trimmed
and soaked for 5-10 min in a fixative solution (10% TCA).
• Strip is then stained by submersion in Ponceau S solution for
10 min.
• De-stained in several changes of acetic acid.
• For densitometry(quantitative measurement of optical density in light-sensitive materials, such
as photographic paper or photographic film, due to exposure to light), the strip may be used
as such or it may be cleared by a dip in clearing solution and
drying in an oven at 60-80°C.
11. o USES:
• Identification of abnormal patterns of plasma proteins in various disease
processes.
• Identification/quantitation of normal and abnormal protein bands.
• Identification and quantitation of normal and abnormal haemoglobins(sickle
cell anemia, anemia & other haemoglobin disorders)
• To find M-proteins (sign of myeloma).
• Quantitation of lipoproteins.
• To separate DNA/RNA.
• Identification of isoenzymes.
Pattern for Serum protein
Electrophoresis in various diseases
