Definition, factors affecting electrophoresis, classification of electrophoresis in general, Iso-electric focusing in detail, IEF and its types (based on ampholytes), step wise procedure of IEF process, Problems involved and their remedies, Capillary iso electric focusing and its types, detection of analytes explained in animation (so watch it in slide show mode), advantages and applications of CIEF.

1. ELECTROPHORESIS
Prepared by,
Ranjith. R
M-Pharma-Ist sem (QA)

2. Definition of Electrophoresis
• Electrophoresis is migration of charged particles or
molecules in a medium under the influence of an applied
electric field.
• Many important biological molecules such as amino acids,
peptides, proteins, nucleotides, nucleic acids possess
ionisable groups and, therefore, at any given pH, exists in
solution as electrically charged species either as cations or
anions.
• Under the charge of an electric field these charged particles
will migrate either to cathode or to anode, depending on
the nature of their net charge.
2

3. The Rate of migration of charged molecules depends
upon following factors:
(a) The strength of electric field.
(b) Relative hydrophobicity of the sample.
(c) Ionic strength and temperature of the buffer.
(d) Molecular size of the sample.
(e) Net charge density of the sample.
(f) Shape of the sample.
In the process of electrophoresis large molecules have
more difficulty in moving through the supporting medium
(i.e., gel) whereas the smaller medium has more mobility
through it. 3

4. why to take a supporting medium?
Supporting medium :- A supporting medium is a physical
support through which the charged molecules get
separated.
It has two primary functions,
- adsorption and
- molecular sieving of the taken molecules which are in-
tended to be separated.
why not to follow the basic principles of
electrochemistry, using an electrophoretic tank with two
electrodes for separating these charged molecules. 4

5. • The pioneering work on electrophoresis by Tiselius et al. in
the year 1948 was performed in free solution only.
problems associated with this approach,
- Current caused by the expansion of a liquid, solid, or gas as its
temperature rises.
- This makes the separated molecules distorted and very ran-
domly arranged
Solution,
• The adverse effects of convection currents could be
minimized by stabilizing the medium. This was achieved by
carrying out electrophoresis on a porous mechanical support.
• Due to its viscosity (which is absent in free solution) the
supporting media cut down the convention currents due to
which we get the separated molecules in sharp zones.
5

6. Classification of
Electrophoresis:
• Depending upon the presence or absence of supporting media the
electrophoresis can be classified as
1. free electrophoresis
- micro-electrophoresis
- moving boundary electrophoresis
2. zone electrophoresis
(a) Paper electrophoresis
(b) Cellulose acetate electrophoresis
(c) Capillary electrophoresis
(d) Gel electrophoresis
(e) Isotachophoresis
(f) Iso electric focusing
3. Special techniques
6

7. Special techniques,
(a) Immuno Electrophoresis
(b) Thin layer electrophoresis
(c) Continuous electrophoresis.
7

8. Free Electrophoresis:
• In this type of electrophoresis a free electrolyte is
taken in place of supporting media. Nowadays this
type of electrophoresis has become out-dated and
mostly used in non-biological experiments.
• It is mostly of two types
- the micro-electrophoresis which is mostly used in
calculation of Zeta potentials (a colloidal property
of cells in a liquid medium) of the cells
- moving boundary electrophoresis which for many
years had been used for quantitative analysis of
complex mixtures of macromolecules, especially
proteins. 8

9. • Zone Electrophoresis:
• This is the most prevalent electrophoretic technique of these days.
In this type of electrophoresis the separation process is carried out
on a stabilizing media.
The zone electrophoresis is of following types;
1. Paper Electrophoresis
2. Cellulose acetate electrophoresis
3. Capillary electrophoresis
4. Gel Electrophoresis
(a) Agarose Gel Electrophoresis
(b) SDS-Page (Sodium Dodecyl Sulphate (SDS) polyacrylamide gel
electrophoresis)
(c) Pulse Field Gel Electrophoresis (PFGE)
(d) Two-Dimensional Electrophoresis, It is carried out in two
dimensions:
• 1st Dimension Separation: isoelectric focusing
• 2nd Dimension Separation: Isoelectric focusing is followed by SDS-
PAGE
9

10. ISO-ELECTRIC FOCUSING
10

11. Common terms to be understood….
• Amphoteric molecules:- Amphoteric molecules contain
ionizable acidic and basic groups and can take a net
positive or negative charge, depending on the pH of the
molecule’s environment.
ex :- Proteins and peptides.
• Carrier ampholytes are aliphatic oligo-amino oligo-
carboxylic acid molecules of varying length and
branching. They can be prepared by Michael reaction of
unsaturated carboxylic acid with polyethylene amine.
Therefore, carrier ampholytes are amphoteric
molecules themselves containing both ionizable acidic
and basic functional groups and have a defined pI.
Carrier ampholytes have average molecular weights
ranging from approximately 200 to 1000 11

12. • When a voltage is applied across a carrier ampholyte
mixture the carrier ampholytes with the lowest pI (and
the most negative charge) move toward the anode, and
the carrier ampholytes with the highest pI (and the
most positive charge) move toward the cathode.
• The other carrier ampholytes align themselves
between the extremes, according to their pIs, and
buffer their environment to the corresponding pH. The
result is a continuous pH gradient.
12
Creating a carrier ampholyte pH gradient.
(A) No voltage applied.
(B) (B) Ampholytes and proteins move by
electrophoresis when charged.
(C) At isoelectric pH, ampholytes and proteins
are focused.

13. • Iso-electric point or pI :- The pH where the
amphoteric molecule becomes neutral i.e., where
positive and negative charges in the molecules are
balanced.
13

14. Isoelectric focusing is an electrophoretic
method in which proteins are separated on the basis
of their pI
• It makes use of the property of proteins that their
net charges are determined by the pH of their local
environments.
• Proteins are +vely charged in solutions at pH values
below their pI. (moves towards cathode)
• Proteins are -vely charged in solutions at pH values
above their pI. (moves towards anode)
14

15. What happens to the sample analyte (Protein) in
presence of electric field in a linear pH gradient field?
• When a protein is placed in medium with a linear pH
gradient and subjected to an electric field, it will
initially move toward the electrode with the opposite
charge.
• During migration through the pH gradient, the protein
will either pick up or lose protons. As it does, its net
charge and mobility will decrease and the protein will
slow down.
• Eventually, the protein will arrive at the point in the pH
gradient equalling its pI.
There, being uncharged, it will stop migrating.
In this way, proteins condense, or focus, into sharp bands in the
pH gradient at their individual, characteristic pI values.
15

16. • The principle: In a pH gradient the sample
components migrate towards the anode or the
cathode to the pH values, where their net
charges are zero: their isoelectric points (pI).
• Should a protein diffuse away from its pI, it
would gain a charge and migrate back: this is
the focusing effect.
16

17. • Focusing is a steady-state mechanism with regard to
pH. Proteins approach their respective pI values at
differing rates but remain relatively fixed at those
pH values for extended periods.
• This type of motion is in contrast to conventional
electrophoresis in which proteins continue to move
through the medium until the electric field is
removed.
17

18. 18

19. IEF AND ITS TYPES (based on type of ampholyte)and
pH gradient system.
• IEF can be run in either a native or a denaturing
mode.
• Native IEF is the more convenient option, as precast
native IEF gels are available in a variety of pH
gradient ranges. This method is also preferred when
native protein is required, as when activity staining
is to be employed. The use of native IEF, however, is
often limited by the fact that many proteins are not
soluble at low ionic strength or have low solubility
close to their isoelectric point.
In these cases, denaturing IEF is employed. 19

20. • Urea is the denaturant of choice, as this uncharged
compound can solubilize many proteins not
otherwise soluble under IEF conditions.
Detergents and reducing agents are often used
in conjunction with urea for more-complete unfolding
and solubilization.
Urea is not stable in aqueous solution, so precast IEF
gels are not manufactured with urea.
• Dried precast gels are a convenient alternative; they
have been cast, rinsed, and dried and can be
rehydrated with urea, carrier ampholytes, and other
additives before use.
20

21. Although useful, carrier ampholytes have some limitations.
Because ,
• The carrier ampholyte–generated gradient is dependent on
the electric field, it breaks down when the field is removed.
• The pH gradients are also susceptible to gradient drift (or
cathodic drift), a phenomenon in which there is a gradual
decrease in pH at the cathodic (–) end of the gel and a
flattening out of the pH at the anodic (+) end.
For this reason it is important to not over-focus the
protein, because cathodic drift will increase over time.
• There can be significant batch-to-batch and company-to-
company variations in the properties of carrier ampholytes,
which limits the reproducibility of focusing experiments.
• Another problem encountered with carrier ampholytes is
their tendency to bind to the sample proteins, which may
alter the migration of the protein and render the separation
of carrier ampholytes from the focused protein difficult. 21

22. Acrylamido buffers,
Acrylamido buffers are an alternative means to form pH
gradients that circumvent most of the limitations of carrier
ampholytes.
• Chemically, they are acrylamide derivatives of simple buffers
and do not exhibit amphoteric behaviour.
• The acrylic function of an acrylamido buffer co-polymerizes
with the gel matrix and, by pouring a gel that incorporates an
appropriate gradient of acrylamido buffers, an immobilized
pH gradient (IPG) is formed. The protein sample can be
applied immediately (no pre-focusing is needed).
• The pH gradient is stable and does not drift in an electric
field.
• Additionally, the gels are not susceptible to cathodic drift,
because the buffers that form the pH gradient are
immobilized within the gel matrix.
22

23. 23
Creating an immobilized pH
gradient.
(A, B, C) A gradient of acrylamido
buffers in an acrylamide solution is
cast into a slab gel that is
crosslinked to a plastic support
film.
(D) The gel is washed to remove
polymerization by products.
(E) The gel is dried for storage.
(F) The pH at any point in the gel is
determined by the mixture of
buffers crosslinked into the gel at
that site.

24. • Acrylamido buffers are available as individual
Immobiline™ species with a specific pK value (or
optimum pH buffering range), suitable for casting
gradients from pH 3–10.
• Because reproducible linear gradients with a slope as
low as 0.01 pH units/cm can separate proteins with pI
differences of 0.001 pH units, the resolution possible
with immobilized pH gradient gels is 10–100 times
greater than that obtained with carrier ampholyte–
based IEF.
With carrier ampholytes, conc of about 2% w/v are
best. Ampholyte conc below 1% w/v often result in
unstable pH gradients. At conc above 3% w/v ampholytes
are difficult to remove from gels and can interfere with
protein staining.
24

25. Gels for IEF
• As an analytical tool IEF is carried out in large pore
polyacrylamide gels (5% T, 3% C) which serve mainly as
anticonvective matrices.
• Polyacrylamide IEF gels are polymerized with an initiator
system including riboflavin for photopolymerization.
• Photochemical initiation of polymerization with a
combination of the 3 compounds riboflavin, ammonium
persulfate, and TEMED, results in more complete
polymerization of IEF gels than does chemical polymerization
in gels containing low-pH ampholytes.
• Suitable initiator conc are 0.015% ammonium persulfate,
0.05% TEMED, and 5mcg/ml riboflacin-5’-phosphate.
• Photochemical polymerization is allowed to continue for 2 hr,
with the second hour under direct lighting from a nearby
fluorescent lamp.
25

26. • IEF is best performed in a flatbed electrophoresis
apparatus.
This type of apparatus allows very effective
cooling, which is necessary due to the high voltages
employed for IEF.
• variety of precast gels for IEF are available, like,
- ready-to-use carrier ampholyte gels,
- dried IPG gels, and
- dried acrylamide gels ready for reswelling in a
mixture of carrier ampholytes and
- any other additives desired, such as detergent and
denaturants.
26

27. Precast IEF gels for Flatbed Electrophoresis System
Ampholine™ PAG plate IEF
• pH 3.5–9.5
• pH 4.0–6.5
• pH 5.5–8.5
• pH 4.0–5.0
Immobiline Dry Plate IEF
• pH 4.0–7.0
• pH 4.2–4.9
• pH 4.5–5.4
• pH 5.0–6.0
• pH 5.6–6.6
27

28. Power conditions and resolution in isoelectric focusing
• The pH gradient and the applied electric field determine the
resolution of an IEF run.
• The difference in pI b/n two resolved adjacent protein IEF
bands (∆pI) is directly proportional to the square root of the
pH gradient and inversely proportional to the square root of
the voltage gradient (field strength) at the position of the
bands
• Thus narrow pH ranges and high applied voltages (small ∆pI)
in IEF.
In addition to the effect on resolution, high electric fields
also result in shortened run times. But high voltages in
electrophoresis are accompanied by large amounts of generated
heat.
Thus, there are limitations on the magnitudes of the
electric fields that can be applied and the ionic strengths of the
solutions used in IEF.
28

29. • Because of their higher surface to volume
ratio, thin gels are better able to dissipate
heat than thick ones and are therefore
capable of higher resolution (high voltage).
• Electric fields used in IEF are generally of the
order of 100 V/cm.
• At focusing, currents drop to nearly zero since
the current carriers have stopped moving by
then.
29

30. Staining of IEF gels:
• IEF gels require special staining procedures: the
proteins have to be efficiently fixed, whereas carrier
ampholytes have to be washed out.
A high detection sensitivity is obtained only with
the following staining procedures:
Coomassie Blue Staining of Agarose gels:
Quick Coomassie Blue Staining of
Polyacrylamide gels:
Silver staining of Ampholine PAG plates:
30

31. General Steps involved in staining process,
1. Fixing,
2. Washing,
3. Staining,
4. Destaining,
5. Impregnating,
6. Drying.
31

32. 1. Fixing,
• In the fixing step, the gel is treated with acid. This
renders the macromolecules in the gel insoluble
and prevents them from diffusing out of the gel
during subsequent staining i.e., usually precipitation
of the proteins occur.
Very commonly TCA (tri-chloro acetic acid) is
used as a fixing agent
2. Washing,
• Substances in the gel that interfere with silver
staining such as buffers, ions, denaturants,
detergents or carrier ampholytes, are washed out of
the gel during this step. 32

33. 3. Staining,
In this step, the solvents intended for staining the
analytes are introduced and staining occurs.
4. Destaining,
In this step, the washing procedure is repeated to
destain the strip from excess of staining components.
5. Impregnation,
In this step, the strip paper containing the
separated protein bands is being treated with 5% glycerol
and 10% acetic acid mixture solution which acts similar to
preservative sheets.
6. Drying,
Finally the obtained strip of the analyte bands is
being air dried which can be stored for a long period of
time.
33

34. Steps involved Coomassie Blue
Staining
Quick
Coomassie Blue
Staining
Silver staining
1. Fixing 30 min in 20%
(w/v) TCA
10 min in 300
mL of 20% TCA
A. Fixing sol. I,
20 % TCA
B. Fixing sol. II,
50 % methanol,
10 % acetic acid
C. Fixing solution
III, 5 %
methanol, 7 %
acetic acid.
D. Fixing
solution IV, 2.5
%glutardialdehy
de

35. 35
2. Washing 2 x 15 min in
200 mL fresh
solutions of 10%
acetic acid, 25%
methanol;
2 min in 300 mL
(mix A.0.2% (w/v)
CuSO4+ 20% acetic
acid and B. 60%
(v/v) methanol 1:1)
Washing takes
place in the
fixing step itself
2.a Drying
place 1 layer of
moistened filter
paper and 3
layers of dry
filter paper on
the gel, a glass
plate, and a 1 kg
weight on top.
Remove
everything after
10 min and
finish drying in
the heating
chamber;
----------- -----------------

36. 3. Staining 10 min in 0.5%
(w/v) Coomassie
R-350 in 10%
acetic acid, 25%
methanol
15 min in 300
mL staining
solution (mix A
and C 1:1) at 50
°C while stirring;
Ammoniacal
silver reagent
4. Destaining in 10% acetic
acid, 25%
methanol till the
background is
clear
15 to 20 min
(mix A and B 1:1)
10 % ethanol, 1
% acetic acid
5. Impregnation ----------- 10 min in 200
mL 5% glycerol,
10% acetic acid;
10 % ethanol, 1
% acetic acid, 15
mL glycerol
6. Drying in the heating
cabinet.
air-dry. air-dry.
36
C: dissolve 1 tablet of Phast Blue R in 400 mL of H2Odist, add 600 mL methanol,
stir 5 to 10 min

37. Native isoelectric focusing
37
The following protocol separates proteins under native conditions over a
range from pH 3.5 to pH 9.5 on an Ampholine PAG plate pH 3.5–9.5
precast IEF gel.

38. • Solutions
38

39. Materials and equipment
• IEF anode solution (1 M H3PO4)
• IEF cathode solution (1 M NaOH)
• Kerosene or light paraffin oil
• Ampholine PAG plate pH 3.5–9.5
• IEF electrode strips
• Multiphor II Flatbed Electrophoresis Unit
• MultiTemp III Thermostatic Circulator
• IEF sample applicator strip
• IEF sample application pieces
• EPS 3501 XL or EPS 3501 high-voltage power supply
(capable of delivering 3 500 V while limiting both current
and power)
39

40. Procedure
Sample preparation
1. Dissolve the sample in water to a concentration of 0.5–10 mg/ml for
Coomassie Blue staining.
Buffers and salts should not be present at a concentration greater
than 50 mM. Samples containing insoluble material should be filtered or
centrifuged before use.
Instrument and gel preparation
2. Connect the Multiphor II to the thermostatic circulator and set the
temperature to 10 ºC.
Turn on the thermostatic circulator 10 min before starting the
analysis.
3. Pipette about 1 ml of kerosene or light paraffin oil onto the cooling
plate of the electrophoresis unit.
4. Remove the Ampholine PAG plate from its package and position it in the
centre of the cooling plate, allowing the kerosene or oil to spread evenly
underneath the gel. Make sure no bubbles are trapped beneath the gel.40

41. 5. Use scissors to cut off the ends of the electrode
strips that protrude beyond the ends of the gel.
6. Soak one IEF electrode strip in IEF anode solution
using approximately 3 ml of solution. Remove excess
solution with blotting paper
7. Repeat step 6 with a second IEF electrode strip
using IEF cathode solution.
41
Soak the electrode strip in anolyte
or catholyte.

42. • 8. Apply the electrode strips over the long edges of
the gel. The electrode strip soaked with the anodic
solution should be toward the anodic (+) side of the
cooling plate
42Applying an electrode strip near the anode.

43. Pre-run the gel (optional)
9. Place the electrode holder and align the electrodes
over the centre of the electrode strips. Lower the
electrode holder so that the electrodes rest on the
electrode strips. Connect the two electrodes to the
base unit and place the lid on the unit.
10. Connect the leads to the power supply. Set the
power supply to 1 500 V, limiting the current at 50 mA
and the power at 30 W. Run the gel for 15 min.
• The voltage should start out between 300–600 V
and rise steadily toward 1 500 V during the pre-run
step.
• Pre-running the gel is optional, but can result in a
higher-quality separation. 43

44. Apply the sample and run the gel
11. Turn off the power supply and disconnect the leads. Take
the lid off the unit and remove the electrode holder.
12. Lay the IEF applicator strip across the gel toward either the
anodic or cathodic edge. Check that the contact between the gel
and the applicator strip is uniform.
• The optimal position for sample application varies with
sample type and must be determined empirically. This is best
done using sample application pieces, which are 5 ´ 10 mm
pads of absorbent material that can be placed in multiple
positions on a single gel
44
Placing sample application
pieces across the gel.

45. 13. Pipette the samples or standards into the wells in the
applicator strip or onto the sample application pieces. Use 5–
20 μl sample volumes
45
Pipetting samples into
the applicator strip
wells.
14. Place the electrode holder on the Multiphor base and align the
electrodes over the centre of the electrode strips. Lower the electrode
holder so that the electrodes rest on the electrode strips. Connect the
two electrodes to the base unit and place the lid on the unit.
Positioning the electrode holder over the
electrode strip.

46. 15. Connect the leads to the power supply. Set the
power supply to 1,500 V, limiting the current at 50 mA
and the power at 30 W. Run the gel for 1.5 h.
16. Turn off the power supply and disconnect the
leads. Take the lid off the unit and remove the
electrode holder.
Lift the gel off the cooling plate and place in a
staining tray and proceed with Coomassie Blue or
silver staining
46

47. Troubleshooting
47

48. 48

49. Capillary iso-electric focusing
Besides on a flat surface, IEF in a gel has also
been executed in a tube.
Relatively wide diameter PTFE or glass capillaries (0.5-
2mm id) have been used.
In here, only low voltages can be used so that thermal
gradients in the gel are minimized. In spite of the low
voltage, extensive cooling is required and separation
takes overnight.
Terms to be understood,
Anolyte, an electrolyte solution with low pH.
Catholyte, an electrolyte solution with high pH. 49

50. One characteristic of electrophoresis in fused
silica capillaries is the phenomenon of electro-
osmotic flow (EOF).
50
In a fused silica
capillary
filled with an
electrolyte solution
an electrical double
layer is formed on
the inner capillary
surface.
As silanol groups in
silica are slightly
acidic, the inner
surface is –very
charged when the
electrolyte solution
has a pH>3

51. 51
These charges are
balanced by ions of
opposite charge from
the electrolyte
solution. Since there
must be
electro-neutrality
over the cross section
of the capillary, there
is a slight excess of
+ve ions close to the
inner wall, of the
fused silica capillary.
When an electric field is applied to the electrolyte solution along the
capillary axis, the excess of cations close to the wall, which are mobile,
are drawn to the electrode of opposite charge dragging along their
solvation shell of water. Consequently a net flow of the liquid phase,
EOF, in the capillary occurs.

52. CIEF is of 2 types,
1. Two step CIEF,
Here, 2 separate or detached process takes place:
a. Arrangement or focusing of analyte samples
b. Detection and isolation of analytes past through
the detector.
2. One step CIEF,
Here, the above 2 processes occur simultaneously
Detection of analytes in both the types occurs by two
ways,
- Hydrodynamic or pressure mobilization
- Chemical mobilization 52

53. 53
+ -
capillary
Anode cathode
Detector
Anolyte Catholyte
1 2 3 4 5 6 7 8 9
Hydrodynamic or pressure mobilization
PRESSURE

54. 54
+ -
capillary
Anode cathode
Detector
Anolyte
(Strong acid)
Acetic acid
(Weak acid)
1 2 3 4 5 6 7 8 9
Chemical mobilization

55. 55
+ -
capillary
Anode cathode
Anodic spacer
Iminodiacetic
acid
Cathodic spacer
L-arginine
Detector
Anolyte
Catholyte
or
Weak acid
1 2 3 4 5 6 7 8 9
Advanced mobilization

56. 56

57. 57

58. 58

59. THANK YOU
59