The presentation describes various types of electrophoresis, their principle

1. Gel Electrophoresis

2. INTRODUCTION
 When charged particles move in an electric field
 Electrophoresis is most commonly used for
biomolecule separation such as DNA, RNA or protein
 May be used as a preparative technique prior to use of
other methods such as RFLP, PCR, cloning, DNA
sequencing, or blotting
Arne Tiselius (Nobel Prize in 1948)

3. Principle
• When we place any charged molecules in
an electric field, they move toward the
positive or negative pole according to the
charge they are having.
• Proteins do not have any net charge
whereas nucleic acids have a negative
charge so they move towards the anode
when electric field is applied
Inherent Factors
• charge of the particles
• molecular weight
• Secondary structures
(i.e., its shape).
External Environment
• pH of solution
• Electric field
• Solution viscosity
• Temperature
Factors Affecting gel Electrophoresis

4. TYPES OF ELECTROPHORESIS

5. VERTICAL
AND HORIZONTAL
ELECTROPHORESIS

6. Variable LOW VOLTAGE
ELECTROPHORESIS
HIGH VOLTAGE
ELECTROPHORESIS
Voltage gradient Voltage gradient approx.
5 V cm -1
Voltage gradient approx.
100 V cm -1
Separation materials Used to separate ionic
substances, sugars,
biological and clinical
specimens for amino
acids and proteins
Small ions deriving from
small peptides and
amino acids

7. Immuno Electrophoresis Source:
https://epgp.inflibnet.ac.in/epgpdata/uploads/epgp_con
tent/S001174BS/P001200/M010899/ET/1479966192P5
M32Jan9.pdf
• Immunoelectrophoresis
• t is a process of a
combination of
immuno-diffusion and
electrophoresis.
• “immunoelectrophoresis
” was first coined by
Grabar and Williams in
1953.
This Photo by Unknown Author is licensed under CC BY

8. Principle of Immunoelectrophoresis
• When an electric current is applied to a
slide layered with gel, the antigen
mixture placed in wells is separated into
individual antigen components
according to their charge and size.
• Following electrophoresis, the
separated antigens are reacted with
specific antisera placed in troughs
parallel to the electrophoretic migration
and diffusion is allowed to occur.
• Antiserum present in the trough moves
toward the antigen components
resulting in the formation of separate
precipitin lines in 18-24 hrs, each
indicating reaction between individual
proteins with its antibody.

9. Immuno Electrophoresis Source:
https://epgp.inflibnet.ac.in/epgpdata/uploads/epgp_con
tent/S001174BS/P001200/M010899/ET/1479966192P5
M32Jan9.pdf
• Immunoelectrophoresis
• Polyacrylamide is gels are the most
commonly used matrices in research
laboratories for separation of proteins
and nucleic acids, respectively.
• It is useful in the clinical
characterization (suspected
monoclonal and polyclonal
gammopathies, abnormal proteins,
such as myeloma proteins in
human serum, medical diagnostic
for under/ over production of
proteins) of human serum protein

10. ISOELECTRIC FOCUSING
Principle
 Isoelectric focusing their intrinsic charge or the
isoelectric point. Isoelectric point of a protein is the pH
at which the protein has no net charge.
 Above its isoelectric point, a protein has a net negative
charge and migrates toward the anode in an electrical
field.
 Below its isoelectric point, the protein is positive and
migrates toward the cathode.
 As it migrates through a gradient of increasing pH,
however, the protein's overall charge will decrease until
the protein reaches the pH region that corresponds to its
pI.
 At this point it has no net charge and so migration ceases
as there is no electrical attraction towards either
electrode).
 As a result, the proteins become focused into sharp
stationary bands with each protein positioned at a point
in the pH gradient corresponding to its pI.

11. • Molecules to be focused are distributed
over a medium that has a pH gradient
created by aliphatic ampholytes.
• IEF can be performed using IPG strips. These
are rehydrated with 250 µl of rehydration
buffer (8 M urea, 2 M thiourea, 2% CHAPS,
DTT 0.003%, and IPG buffer, pH 3-10, 0.5%).
• Samples are applied by cup loading and IEF
is performed as per the program.
• IEF is stopped when a total volt-hours (VhT)
is achieved.
• Temperature is set at 20˚C.
• Strips are covered with cover fluid
throughout the run period.

14. IPG STRIPS

15. Second Dimension Electrophoresis (SDS PAGE)
Gel Image after 2DE

16. Polyacrylamide gel
electrophoresis

17. • Polyacrylamide gel is the most useful
and widely used techniques for the
separation and characterization of
nucleic acids and proteins.
• advantages like : it is chemically
inert
• having superior resolution
• stable over a wide range of pH
• wide range of gel can be prepared
by using polyacrylamide gel
• have good temperature and ionic
strength.

18. In PAGE electrophoresis the separation
depends on the friction of the protein within
the matrix and the charge of the given protein
as given in the formula:
Mobility = 𝑉𝑜𝑙𝑡𝑎𝑔𝑒 𝑎𝑝𝑝𝑙𝑖𝑒𝑑 𝑥(𝑡𝑜𝑡𝑎𝑙 𝑐h𝑎𝑟𝑔𝑒 𝑜
𝑛 𝑡h𝑒 𝑝𝑟𝑜𝑡𝑒𝑖𝑛)𝐹𝑟𝑖𝑐𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡h𝑒 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒

19. % Acrylamide in
resolving gel
Effective separation
range (Da)
7.5
10
12
15
20
45,000-200,000
20,000-200,000
14,000-70,000
5,000-70,000
5,000-45,000
Table-2 The various concentration of acrylamide gel
concentration for the effective separation range of proteins
 The pore size of the
polyacrylamide gel can be
modified by changing the
concentration of
acrylamide gel.
 For larger pore size the
amount of monomer can
be decreased
 for smaller pore size gel the
concentration of monomer
or cross linker can be
increased.

20. Apparatus of
polyacrylamide
gel
electrophoresis

21. Buffer systems
 pH may be ranged from 3-10.
 To reduce the heat production
during electrophoresis to a
minimum the ionic strength of the
buffers kept low (0.01-0.1M).
Depending on the
different types of buffer
Native continuous polacrylamide gel
electrophoresis
SDS-
polyacrylamide
gel
electrophoresis
(reducing)
SDS-
polyacrylamide
gel
electrophoresis
(non reducing)
Native
discontinuous
polyacrylamide
gel
electrophoresis

22. •proteins are heated with
anionic detergent SDS or
SLS to dissociate into their
constituents subunits.
•Separation based on size of
the polypeptide.
• Protein treated
with an excess
of SDS and
soluble thiol.
•buffers ions used in the
gel and in the electrode
reservoir are different
and it contains two
types of gels i.e large
pore stacking gel and
small pore gel (different
ionic strength and pH)
• the buffer ions
and pH used for
gel, electrodes
and throughout
the sample are
same Native
continuous
polyacrylamide
gel
electrophoresis
Native
discontinuous
polyacrylamide
gel
electrophoresis
SDS-
polyacrylamide
gel
electrophoresis
(Non reducing)
SDS-
polyacrylamide
gel
electrophoresis
(reducing)

23. SDS-polyacrylamide gel electrophoresis (Non reducing)

24. High pH buffer
systems
• i) Stacking gel buffer (Tris –HCl, pH 6.8)
• ii) Resolving gel buffer (Tris –HCl, pH 8.8)
• iii) Reservoir buffer (Tris-glycine, pH 8.3)
Natural pH buffer
system
• i) Stacking gel buffer (Tris-phosphate, pH 5.5)
• Ii) Resolving gel buffer(Tris-HCl, pH 7.5)
• ii) Reservoir buffer(Tris ethyl barbiturate, pH 7.0)
Low pH buffer
systems
• i) Stacking gel buffer (Acetic acid-KOH, pH 6.8)
• ii) Resolving gel buffer (Acetic acid-KOH, pH 4.3)
• iii) Reservoir buffer (Acetic acid-α alanine, pH 4.5)
Commonly used buffers for native discontinuous polyacrylamide gel electrophoretic systems

25. Fig. SDS-PAGE electrophoresis (reducing)

26. Gel staining: Coomassie blue
Chemical reagents required:
Brilliant Blue R-250 (BBR)
Fixing solution:
ratios of
methanol(50),
acetic acid(10)
and water(40)
Destaining
solution:
methano(45)l,
acetic acid(10)
and water(45).

27. Silver staining Protocol
Destaining Protocol
Destain until no band is visible
Gel is washed 3-5 times for 10 min in a distilled water which
should be sterile also till all the stain is removed

28. Capillary electrophoresis

29. INTRODUCTION
 introduced in late 1800’s employing experimentation with the help of glass U tubes.
 Arnes Tiselius (1930) for the separation of proteins
 Hjerten (1960’s) for the use of capillary
 Jorgenson and Lukacs for the separations of both inorganic and organic compounds.
 The replacement of U tube with capillary having thin dimensions enhanced the surface to volume ratio and
increased the efficiency making the separation better.
 The capillary electrophoresis uses an electric field for the separation of components of a mixture within the
narrow dimensions of a tube

30. PRINCIPLE
 modern high performance separation
method.
 The separation is based on different
migration of analytes in a capillary
over which a high voltage (typically
10-30 kV) is applied. Typical inner
diameters of commonly used
capillaries are in the range of 25-75
μm
 Electrically charged particles are
moving in the applied electric field in
a direction determined by their
charge and the field orientation.
background electrolyte (BGE) (isocratic),

31. INSTRUMENTATION
fused silica (with
polyimide coating)
having 10-100 micro
meter inner diameter
and 40-100 cm length
electrophoregram

32. Modes of separation in capillary electrophoresis
CE modes Separation mechanism Application
Capillary zone eletrophoresis
(CZE)
Separation is based on mobility
differences of analytes in an electric
field based on on the size and charge
to mass ratio of analyte ions.
Charged molecules
Capillary gel electrophoresis
(CGE)
Mechanism is based on the solute
size as the capillary is filled with a gel
or polymer network that inhibits the
passage of larger molecules
Macromolecules such as protein and
DNA
Micellar electrokinetic
chromatography
(MEKC)
Separation mechanism is based on
the differential partition of the
solutes between the hydrophobic
interior of a charged micelle and the
aqueous phase
Neutral and charged molecules

34. Modes of separation in capillary electrophoresis
CE modes Separation mechanism Application
Capillary electrochromatography
(CEC)
Capillary is packed with a stationary
phase that can be capable of
retaining solutes in a manner similar
to column chromatography.
Neutral and charged moleclues
Capillary isoelectric focusing
(CIEF)
Analytes are separated on the basis of
their isoelectric points
Zwitterionic
Capillary isotachophoresis
(CITP)
Sample zone migrate between a
leading electrolyte at the front and a
trailing electrolyte at the end. All of
solutes travel at the same velocity
through the capillary but are
separated on basis of differences in
their mobilities.
protein and peptide
Anions or cations

35. Detectors used in Capillary Electrophoresis
UV-
visible
absorptio
n
detection
aTeflon coated or polyamide
coated capillary acts as
reference cell.
Fluoresce
nce
emission
detection
fast response and improved
limits of detection
set-up for fluorescence
detection in a capillary
electrophoresis system is
complicated.
Mass
spectrosc
opy
detection
Other
detectors
refractive index detectors,
amerometry,
conductometry and
potentiometry.
 The detector response in the
end of the capillary (the
electrophoregram), has a
characteristic peak profile that
is a function of many factors
(type of sample, injection,
detection, sorption, mobility
differences, etc.).
 The qualitative characteristics
are related to the migration
time of the peaks and the
quantitative characteristics are
represented by the peak height
or the peak area.

36. Applications of CE
Inorganic analysis involves
derivatization of ions using
organic chelators like
cyanide, lactate, EDTA, and
a-hydroxyisobutyric acid.
Organic analysis: analysis of two retinoic acid
isomers and their degradation products,
dihydroxybenzoic acids, aromatic sulfonic
acids, catecholamine metabolites, mixture of
oxalate, tartrate, malate, succinate, lactate,
acetate.

37. Dye Analysis
analyses of anionic dyes and cationic
intermediates with the help of borate-SDS
buffers.
Food and Agriculture analysis
analysis of essential oil extracts, pungent food
components,etc.

38. Gel documentation systems
• Gel documentation systems, also known as
'gel docs' or 'gel imagers,' are used to
record and analyze the results of gel
electrophoresis and membrane blotting
experiments.
• These instruments are necessary for
visualizing stained or labeled nucleic acids
and proteins in media such as agarose,
acrylamide, or cellulose and supported
stains
• Systems come in a variety of
configurations depending on application,
throughput, and sample type.

39. https://www.bio-
rad.com/webroot/web/pdf/lsr/lit
erature/Bulletin_7284.pdf

41. http://dfs.nic.in/pdfs/In%20House%20ca
libration_Merged-1.pdf

42. Important reading material
• Annexure – 7: Standard operating procedure for calibration of UV-Visible
Spectrophotometer
• Annexure – 9: Standard operating procedure for calibration of High
Performance Liquid Chromatography (HPLC) System
• Annexure – 10: Standard operating procedure for calibration of Gas
Chromatograph-Flame Ionization (GC-FID) System
• Annexure – 11: Standard operating procedure for calibration of Gas
Chromatograph-Mass Spectrometry (GC-MS) System
• Annexure – 14: Standard operating procedure for calibration of Ion
Chromatography (IC) System
• Annexure – 15: Standard operating procedure for calibration of High
Performance – Thin Layer Chromatography (HPTLC) System