Electrophoresis

Electrophoresis
Electrophoresis is a seperative procedures.
Electrophoresis is the name given to the
movement of charged particles through an
electrolyte subjected to an electrical field.
If these are differently charged they will move in
opposite directions , the positively charged to the
cathode and the negatively charged to the anode.
The rate of migration of particles of like charge
will depend , among other things , on the number
of charges each carries.
Different rates of migration separate a complex
mixture such as plasma proteins into a number of
fractions of like mobility , the sharpness of
separation depending upon the extent to which
each fraction is homogeneous in its mobility.
 Factors influencing the rate
of migration
(1)Support media.
(2)Composition and concentration of buffer.
(3)Voltage , current and heating effect.
(4)Size and shape of particles.
Requirement of
Electrophoresis technique
(1)Support media
Cellulose acetate have an advantages of over
paper:-
(ii)Albumin trailing.
(iii)Time required to complete the separation 60
minutes
(iv)Easy to remove excess day.
(iV)Cellulose acetate can be scanned.
(2)Tank
(3)Power supply
 (4)Buffer
(i)Tris borate EDTA(TBE).
(ii)Barbitone Buffer.

(5)Dye with fixatives

Ponceau S dye+TCA.

(6)Washing solution
(i)Methanol.
(ii)Ethanol.
(iii)Glacial acetic acid.
 Electrophortic patterns of
plasma proteins in normal
Should be performed on serum from clotted
and
blood , because disease
the presence of fibrinogen may
mask , or be interpreted as , an abnormal band or
protein.
Normal pattern
Acute infection
Chronic infection
Nephrotic syndrome
Multiple myloma
Hepatic cirrhosis
Alpha -1-Antitrypsin deficiency
Immunoglobulin deficiency
 Haemoglobin
electrophoresis
Steps of haemoglobin
(A)Blood collected into EDTA.
electrophrosis
(B)Centrifuge & remove the plasma.
(C)Preparation of haemolysat(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.
 Urine
The urine electrophoresis
sample needs to be concentrated due
to the fact that there is usually a small amount of
protein in urine.
Technique have been used to concentrate urine
by using semipermeable membrane.
(i)Immerse the membrance in ethylene glycol.
(ii)The ethylene glycol drain the water from the
membrane.
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.
 Ultracentrifugation
This method allows separation according to
density and also yield four fractions.