2. • Electrophoresis is a technique used to
separate and sometimes purify
macromolecules - especially proteins and
nucleic acids - that differ in size, charge
• As such, it is one of the most widely-used
techniques in Hematology ,biochemistry and
molecular biology
3. • When charged molecules are placed in an
electric field, they migrate toward either the
positive or negative pole according to their
charge. In contrast to proteins, which can have
either a net positive or net negative charge,
nucleic acids have a consistent negative
charge imparted by their phosphate
backbone, and migrate toward the anode.
4. Electrophoresis is carried out by applying a thin layer
Solid matrix with pores which are used:
• paper
• starch
• cellulose acetate
• polyacrylamide
• agar/agarose
Molecules in the sample move through porous matrix at
different velocity.
5.
6.
7. Haemoglobin Electrophoresis
• Haemoglobin Electrophoresis is a method of
determining the type and quantity of
haemoglobin in blood, by observing the rates
of transit of these negatively-charged proteins
in an electric field medium. It is used to
diagnose so-called haemoglobinopathies
8. • The most common types of normal hemoglobin are:
• Hemoglobin A.(α2β 2)This is the most common type of
hemoglobin found normally in adults
• Hemoglobin F (fetal hemoglobin (α2γ2 )This type is
normally found in fetuses and newborn babies.
Hemoglobin F is replaced by hemoglobin A (adult
hemoglobin).
• It is increased in Thalassemia Major
• Hemoglobin A2(α2δ2) This is a normal type of
hemoglobin found in small amounts in adults.
• It is increased in Thalassemia Minor
9. Abnormal hemoglobin. 1
• There are more than 350 types of abnormal
hemoglobin. 1 The most common are:
• Hemoglobin S. This type of hemoglobin is present
in sickle cell disease.
• Hemoglobin C. This type of hemoglobin does not
carry oxygen well.( Hemoglobin C disease)
• Hemoglobin E. This type of hemoglobin is found
in people of Southeast Asian descent.(bengali)
• Hemoglobin D..(Punjabi)
10. Normal Haemoglobin
• Hb A: 95% to 98%
• Hb A2: 2% to 3%
• Hb F: 0.8% to 2%
• Hb S: 0%
• Hb C: 0%
• In infants and children, these hemoglobin molecules
make up the following percentages of total
hemoglobin:
• Hb F (newborn): 50% to 80%
• Hb F (6 months): 8%
• Hb F (over 6 months): 1% to 2%
11. Electrophoresis is carried out by applying a thin layer
Solid matrix with pores which are used:
• paper
• starch
• cellulose acetate
• polyacrylamide
• agar/agarose
Molecules in the sample move through porous matrix at
different velocity.
12. Cellulose acetate electrophoresis
• Cellulose acetate is an acetate salt of cellulose
produced by treating cotton with acetic acid using
sulphuric acid as a catalyst.
• Migration takes place on the buffer film on the surface
of the cellulose acetate plate or membrane.
• Cellulose acetate electrophoresis may be used for
qualitative identification of variants, but also with
elution for quantization of the haemoglobins, A2, A, S,
D, Lepore, α-chain variants, Hb H and Hb Bart's.
13. Reagents and materials
• Tris-EDTA Boric Acid (TEB) buffer, pH 8.4.
-Tris hydroxymethyl amino methane (TRIS): 10.2 g
-Ethylene diamine tetracetic acid (EDTA): 0.6 g
-Boric Acid: 3.2 g
-Make up to 1 litre with distilled water
• Whatman No. 3 chromatography paper.
• Cellulose acetate membranes made by Sartorius and by Shandon
• HbA2 control, as supplied by the National Institute for Biological
Standards and Control NIBSC.
• The control has been produced by freeze drying a solution of
haemoglobin prepared from human cells and made stable by the
addition of sucrose (200 mM), potassium cyanide (6mM) and
chloramphenicol (1 mg/dl). Reagent with an assigned value of 5.3% of
total haemoglobins present.
15. Equipments
• Power supply capable of delivering a constant
current
• An horizontal electrophoresis tank with
adjustable bridge gaps and a polarity indicator
(eg. Shandon).
• Roller mixer.
• Spectrophotometer
16. Haemolysate preparation
• Collect blood in 5 ml blood in EDTA vial
• Wash RBC three times
• Lyse 2 volumes of washed packed RBC in one
Volume of distt. water and then add CCl4
Or lyse By freezing and thawing the add CCL4.
• Shake the tubes vigorously for approxim for 1 mt.
• Centrifuge at 3000 rev/minute) for 30 mts.
• Transfer the supernatant to a clean tube adjust
• Hb to 10 gms/dl
17. Method
• The electrophoresis tank is prepared by filling
the tank with 900ml approximately of TEB
buffer wicks are cut from Grade No. 3
chromatography paper and were placed along
the 22 cm long bridges in the tank.
18. • The cellulose acetate membranes are cut in 40xl00 mm each and
soaked (shiny side down) in TEB buffer for 5 minutes. Five strips are
plotted and placed on the electrophoresis tanks.
• Voltage current is applied at 250 V for 5 minutes to the membranes
to equilibrate the membranes with the buffer.
• The current is turned off and 5-10 μl haemolysate (10 g/dl) is
applied on each membrane at the cathodal end using a capillary
tube.
• Then the voltage is set at 250-300 V working at constant current of
2 mA for each strip.
• The electrophoresis is run for approximately 45 minutes to one
hour until there is a clear area between the bands.
19. • After 25 mts electrophoresis,immediately
transfer cellulose acetate membrane to
• Ponceau S for 5 min.
• Remove excess stain by washing for 5 min. in
First acetic acid reservoir and for 10 mts in
each of remaining two.
20. • The current is then turned off and the separated
HbA2 on the cellulose acetate membrane is cut
and immersed in a tube containing 4 ml of
distilled water and the HbA in a tube containing
16ml distilled water. If a haemoglobin variant is
present then this is cut separately into 4 or 16 ml
distilled water depending on the quantity of the
variant present.
• Note that a blank is prepared from the same run
by cutting a piece of clear cellulose acetate strip
that was immersed in 4ml distilled water.
21. • The tubes are then placed on a roller mixer for
30 minutes for the haemoglobin elution.
• The strips are removed and the tubes are then
centrifuged for 10 minutes at 3.000 rpm.
• The absorbance of each haemoglobin is read
at 413 nm against the blank on a
spectrophotometer.
22. • The results are calculated as follows:
Absorbance of HbA2 x 100
• % of HbA2 = ---------------------------------------
Abs. of Total (HbA x 4) + absorbance of
HbA2
23. • The haemoglobins migrate on the cellulose
acetate membrane from cathode to anode in
the following order: HbA2, Hb E, Hb C, Hb D,
Hb S, Hb Lepore, Hb F, Hb A and the fast
moving haemoglobins Bart's and Hb H
24. Cellulose acetate electrophoresis
Jump to: navigation, search
Cellulose acetate is an acetate salt of cellulose
produced by treating cotton with acetic acid using
sulphuric acid as a catalyst. Migration takes place on
the buffer film on the surface of the cellulose acetate
plate or membrane. Separation of the proteins is
primarily by charge. Cellulose acetate electrophoresis
may be used for qualitative identification of variants,
but also with elution for quantitation of the
haemoglobins, A2, A, S, D, Lepore, α-chain variants,
Hb H and Hb Bart's.
25. • Electrophoresis can be one
dimensional (i.e. one plane of
separation) or two dimensional.
• One dimensional electrophoresis is
used for most routine protein and
nucleic acid separations. Two
dimensional separation of proteins is
used for finger printing , and when
properly constructed can be
extremely accurate in resolving all of
the proteins present within a cell
(greater than 1,500).
• Most common stabilizing media are
polyacrylamide or agarose gels.
26. Support media:
1. Filter paper: paper electrophoresis (Principle same
as paper electrophoresis)
Moist paper is placed on a supporting rack, samples
are applied with a capillary applicator, paper is
loaded onto the apparatus with its ends dipping
into an electrode vessel containing buffer, and
covered to prevent dryingat the end of run, paper is
carefully taken out and
dried, and examined by
appropriate detection
technique
27. Disadvantage
• Thin paper: Tear
• Thick paper: Boundaries between the zones distort
• OH group of cellulose interact with biomolecules and
results in ‘tailing’
• water absorbing and electrical conductivity capacity of
paper depends on its content (96% -cellulose)
• Electro osmosis
28. Electro osmosis
• dissociation of COOH group(cellulose matrix)
generation of (H3O)+ ionMoves (electric field)
• A correction factor for electrophoretic mobility:
• distance between the sample origin and the position
of the blue dextran (does not ionize)
• 20V cm-1 – zone diffusion of small molecules
• 200 V cm-1 – High voltage EP
29. 2. Cellulose Acetate Membrane: Advantages over paper
• OH groups of cellulose are esterified by acetylation
• spectrophotometric determination:Use of mineral
oil of RI equals to that of the membrane;
• Easy dissolution: Recovery of separated sample
• Procedure similar to paper
30. 3. Gel Media:
Network of polymer molecules
surrounded and penetrated by
buffer
space between the gel molecules
are the porous
frictional resistance to movement
(1) relative size of gel pore
(2)radius of the molecule;
exerts a sieving effect
31. a) Starch Gels:
• earlier work with proteins used starch as support
• cheap, easy and reasonably quick
Disadvantage
• pore size can vary within only a narrow range
(determined by the starch conc)
• contains some negatively charged side-chain:
hinder separation
32. b) Polyacrylamide Gels:
Made by cross linking acrylamide with N, N’-
methylenebisacrylamide
Requires an initiator (APS of PPS) for chemical
polymerization & light and ribofalvin for
photopolymerization
Crosslinking reagent is N, N, N’, N’-
tetramethylethylenediamine (TEMED)
33. Ratio of cross-linking agent to acrylamide is a
factor determines the pore size of the gel
Can be tube gel or slab gel
1. Tube Gel
34. 2. Slab Gel EP: Advantage; heat dissipation is better,
higher resolving power; can be used horizontally
(when gel conc is low) or vertically
35. c) Agarose Gel:
Agarose, obtain from seaweeds, linear polymer of D-
Gal and 3,6-anhydroGal
agarose dissolved in boiling water when cooled
forms a gel (held by H-bonds),
pore size relatively large: large proteins or nucleic
acids (too large to enter PAG)
Contaminants charge groups (sulphate and
carboxylic acid ) interfere with separation
36. Factors affecting electrophoretic mobility:
Number of parameter of electrophoretic system:
i) for zone EP, type of support medium chosen,
and if it is a gel, its pore size
ii) pH of the electrophoresis buffer
iii) ionic composition of buffer
iv) applied voltage
v) temperature
37. USES OF ELECTROPHORESIS
1. DNA analysis
Forensic investigation
Paternity tests
2. protein analysis
Of blood or urine samples
3. antibiotic analysis
Synthesis new antibiotic
Analyse which type of bacteria is antibiotic resistant
4. Vaccine analysis.
