Electrophoresis is a separation  technique that is based on the movement of charged particles in an electric field. Electrophoresis is an analytical method of separating charged particles based on their relative mobilities in an electric field

1. Electrophoretic Techniques for
Life Science Researchers
Presented by
Dr. B. Victor., Ph.D.,
email : bonfiliusvictor@gmail.com
blog : bonvictor.blogspot.com

2. Presentation out line
• Origin definition and historical notes
• Electromigration, electromotive force and
electrophoretic theory.
• Types of electrophoretic methods.
• Buffers and support media.
• Frontal, zonal, starch and agarose gel
electrophoresis.
• Modifications of gel systems.
• SDS-PAGE and DISC- PAGE systems.
• Practical applications.

3. What is electrophoresis?
Electrophoresis
• Electrophoresis is
a separation technique that is
based on the movement of
charged particles in an electric
field.

4. Meaning of electrophoresis
electrophoresis
• The term „electrophoresis‟ was
coined from the Greek word
‘phoresis‟, which means „being
carried‟.
• Electrophorosis literally means
„to carry with electricity‟.

5. Historical notes
In 1879, Hermann von In 1930, Swedish
In 1807,Russian chemist Arne Tiselius
Helmholtz generalized
physicist Alexander described the first
the experimental
Reuss identified the electrophoretic system.
observations into an
migration of colloidal
equation of He received the Nobel
particles in an electrical
electrophoretic prize in chemistry in
field.
principles. 1948 for his work.

6. Definition of electrophoresis
Micro -bioanalysis
• Electrophoresis is an
analytical method of
separating charged
particles based on their
relative mobilities in an
electric field.

7. Biomolecules with ionizable groups
Amino acids
Peptides DNA
proteins RNA
nucleotides

8. Electro-migration
At any given pH , the electrically
charged molecules may exist in solution
either as cations (+) or anions (-).
Negatively Positively
charged charged
molecules move molecules move
to the anode(+). to the cathode(-).

9. Transport Processes in
electrophoresis
• Electro migration
• Negatively charged molecules (anions) move towards
the anode (+).
• Positively charged molecules (cations) move towards
the cathode (-).
• Highly charged molecules move faster towards the
electrode of opposite charge than those with lesser
Electro osmosis
• movement of entire fluid near wall of capillary in one
direction!
• anode (+ve) -> cathode (-ve)

10. Electromotive force(EMF)
• Electrophoresis is based on electromotive
force(EMF) that is used to push or pull the
molecules through the gel matrix.
• By placing the mixture of molecules in wells in
the gel and applying an electric current, the
molecules will migrate through the matrix.
• The separated molecules take directions based
on the total electric charges.

11. Principle of velocity of migration of
separated molecules
Velocity of migration of the molecules, v=E.q / f
Where
E=electric field in volts/cm
Q=the total electric charge on the molecule
F=the frictional coefficient which is a friction of
the mass and shape of the molecule.

12. Electrophoretic Theory
• Two laws are relevant to the use of power
supplies for electrophoresis of macromolecules:
• Ohm’s Law and
• Second Law of electrophoresis.

13. Ohm’s Law
• Current (I)=Voltage (V)/Resistance (R)
• Ohm‟s Law states that current is directly
proportional to the voltage and is inversely
proportional to the resistance.
• Resistance of the system is determined by the
buffers used, the type and configurations of the
gels being run, and the total volume of all the
gels being run.

14. Second Law
• Watts (W)=Current (I) x Voltage (V)
• The Second Law states that power or watts (a
measure of the heat produced) is equal to the
product of the current and voltage.
• Since V=I x R, this can also be written as
Watts=I2 x R.

15. Factors influencing rate of
migration of ions
Net charge of the molecule
Size and shape of the molecule
Buffer pH
Strength of electrical field
Properties of support media
Temperature of the operating system

16. Electrophoresis apparatus
Power pack Buffers
Electrophoresis Electrophoresis
Equipment media
Electrophoresis
support media
unit
Electrophoretic
chamber

17. Types of electrophoresis
Paper
electrophoresis
Disc
electrophoresis
Moving boundary/frontal
electrophoresis
Column
electrophoresis
Zonal
Electrophoresis electrophoresis
Capillary
electrophoresis
Immuno-electrophoresis
Open block
electrophoresis
Isoelectric
focusing

18. Gel electrophoresis
Gel electrophoresis
Tube gel Slab gel
electrophoresis electrophoresis
vertical slab gel Horizontal slab gel
electrophoresis electrophoresis

19. Buffers
Barbitone buffer – • serum protein separation ,
(around 8.0 pH) • poor resolution, weak buffer.
Phosphate buffer- • Enzyme separation,
( around 7.0 pH) • low buffering capacity.- high conductivity
Tris – borate – EDTA • Nucleic acid Separation
buffer (TBE) -(pH • Good resolution , high buffering capacity , low
around 8.0) conductivity.
Tris – acetate – EDTA • Nucleic acid separation
buffer (TAE)- (pH • high resolution , high buffering capacity ,
around 8.0) low conductivity.
• protein separation
• high
Tris – glycine buffer - • Protein separation
(pH more than 8.0)- • high buffering capacity , low conductivity

20. Support media
• Paper – poor conductor of electricity absorbate proteins,
non - transparent poor resolution.
• Agar- flow of solvent electro endosmosis, vary thickness ,
transparent poor resolution
• Cellulose acetate strip- tailing of bands poor resolution
non-absorbing.
• Starch- form opaque gels non-absorbing high resolution
• Agarose -highly transparent porous – high resolution east
preparation
• Acrylamide – stable , non –reactive highly transparent.

21. Moving boundary/frontal electrophoresis
• Protein solution is injected Tiselius in 1937 introduced
into a quartz U-tube. this technique.
• The arms of the U- tube are
filled with buffers.
• When electricity is passed,
different species of protein
molecules separate in to
bands of proteins.
• Discrete zones does not
occur.

22. Zonal electrophoresis
• Sample is applied as a narrow
band.
Consden,Gordon and Martin in
• Separation occurs discrete bands. 1946 introduced this technique
• Numerous support media –
paper, cellulose acetate, agar gel
starch gel and acrylamide gel can
be used

23. Paper electrophoresis
• A small volume of the This technique was introduced
by Durrum (1950), Flynn and
sample is placed evenly Mayo (1951)
along a line drawn across a
strip of Whatmann paper
previously soaked in buffer.
• The ends of the paper are
soaked in buffer solutions.
• Passage of electricity cause
separation.

24. • Starch matrix is suitable for
isoenzymes . Starch gel
• Partially hydrolysed potato starch electrophoresis
is used.
• The gels are slightly more opaque
than acrylamide or agarose.
• Non-denatured proteins can be
separated according to charge
and size.
• They are visualised using Napthal
Black or Amido Black staining.

25. Cellulose acetate
electrophoresis
• Strips of cellulose acetate are Kohn (1957-1961) introduced this
technique
used.
• Better resolving power
• No absorption of proteins
• No trailing
• Excellent separation of
plasma proteins.
• transparent

26. Gel electrophoresis that involves the use of a
gelatinous material such as agarose, acrylamide,
starch or cellulose acetate as the matrix.
• The gel acts as a support medium for the sample. Gel electrophoresis
• Gels are used to separate samples containing
proteins or DNA. Electrophoresis through agarose or
polyacrylamide gels is a standard
Starch Gel -- swollen potato starch granules. method used to separate, identify and
Agarose Gel is a natural linear polymer extracted purify nucleic acids.
from seaweed that forms a gel matrix by hydrogen-
bonding when heated in a buffer and allowed to
cool.
Polyacrylamide Gels -Polyacrylamide gel is
made chemically by acrylamide (the monomer) and
bisacrylamide (the cross-linker) catalyzed by
initiator (amonnium persulfate or riboflavin) and
accelerator (TEMED).
Acrylamide can be polymerized into any
desired shape :
• Tube Gels -- polymerize in glass tubing ==>
cylindrical shape
• Slab Gels -- polymerize between glass plate

27. Uses of gel electrophoresis
• Human DNA can be analyzed to provide evidence in
criminal cases, to diagnose genetic diseases, and to solve
paternity cases.
• Samples can be obtained from any DNA-containing
tissue or body fluid, including cheek cells, blood, skin,
hair, and semen.
• A person‟s “DNA fingerprint” or “DNA profile” is
constructed by using gel electrophoresis to separate the
DNA fragments from several of its highly variable
regions.
• Conservation biologists use DNA profiling to determine
genetic similarity and kinship among populations or
individuals.

28. • Agarose gel electrophoresis is a
powerful separation method Agarose gel electrophoresis
frequently used to analyze DNA
fragments generated by restriction
enzymes.
• The separation medium is a gel
made from agarose, which is a
polysaccharide derivative of agar.
• The agarose gel consists of
microscopic pores that act as a
molecular sieve which separates
molecules based upon charge, size
and shape.
• These characteristics,together with
buffer conditions, gel concentrations
and voltage, affect the mobility of
molecules in gels.

29. Differences between agarose and
polyacrylamide gels
Agarose gel Polyacrylamide gel
A polysaccharide extracted A cross-linked polymer of
from sea weed. acrylamide.
Gel casted horizontally Gel casted vertically
Non-toxic Potent neuro-toxic
Separate large molecules Separate small molecules
Commonly used for DNA Used for DNA or protein
separations separations.
Staining can be done before Staining can be done after
or pouring the gel pouring the gel.

30. Modifications of gel systems
Homogeneous • The porosity/percentage of the gel through out
the gel system remains same.
gel system • Only small molecules can be separated.
• It is employed in DISC-PAGE.
Heterogeneous • Two different buffers can be used.
gel system • All types of molecules can be separated.
Gradient • Linear or exponential gradients can be made.
• The formation of gradients can be examined by
Gel system using dyes.

31. Gel electrophoresis
SDS-PAGE
Gel
electrophoresis
DISC-PAGE

32. Sodium dodecyl sulfate-Polyacrylamide
Gel Electrophoresis – SDS- PAGE -1
• SDS-PAGE is a most widely used technique for
analysis and characterization of proteins and
nucleic acids.
• Sample preparation – The protein sample is
heated at 1000C in a dilute solution sodium
dodecyl sulfate .This breaks down all native
quaternary, tertiary, and secondary structures.
• Then b-mercapto ethanol is added to cleave the
disulfide bonds.

33. Sodium dodecyl sulfate-Polyacrylamide
Gel Electrophoresis – SDS- PAGE - 2
• Gel preparation – the polymerization is initiated
by ammonium per sulfate or riboflavin. N-
tetramethyl ethylene diamine (TEMED)
catalyses the formation of free radicals from
persulfate which in turn initiate polymerization.
• Gels ranging from 3 to 30% acrylamide
concentration can be made and can be used for
the separation of molecules up to 1x106 datons.

34. Apparatus setup for slab gel
electrophoresis
Sample wells Cathode
Reservoir
Buffer
Gel
Anode
Reservoir
Buffer

35. Sodium dodecyl sulfate-Polyacrylamide
Gel Electrophoresis – SDS- PAGE - 3
• Sample application –about 2 µg of the sample is
loaded in each well. Over loading of samples
decrease the resolution of bands.
• Marker dyes – to follow the sample tracking a
marker dye e.g. bromophenol blue gives color.
After run the gel was stained with the dye
coomasie blue and photographed.

36. Advantages of polyacrylamide gels
Stable over a wide
Hydrophilic and
Transparent to range of pH,
Chemically inert electrically
light temperature, and
neutral
ionic strength.
Available in wide
Never bind to Superior
range of pore
proteins. resolution
sizes.

37. Applications of PAGE
1. PAGE is used to estimate molecular weight of
proteins and nucleic acids.
2. PAGE is used to determine the subunit
structure of proteins.
3. PAGE is used to purify isolated proteins.
4. PAGE is used to investigate various liver and
kidney diseases by analyzing human serum
proteins.
5. PAGE is used to monitor the changes in
protein content in body fluids.

38. Continuous - discontinuous
gel systems
Continuous system--gel
and tank buffers are the same,
single phase gel; examples are
PAGE, agarose, and starch gels.
Discontinuous system--
gel and tank buffers are different,
two phase gel (stacking gel);
example is PAGE.

39. Discontinuous polyacrylamide gel
electrophoresis –DISC-PAGE - 1
• Two gel systems – a stacking gel and a running
gel
• Several buffer systems-
Cathode - Tris – glycine 8.6 pH
Wells – Tris – Cl 6.5 pH
Stacking gel – Tris – Cl 6.5 pH
Separating gel – Tris – Cl 8.7 pH
Anode - tris – glycine 8.
• Generation of voltage discontinuity

40. Apparatus setup for DISC - PAGE
Cathode
Tris – glycine pH 8.3 Buffer Tank
Sample in Tris -Hcl , pH 6.7
Stocking gel-Tris –Hcl , pH 6.7
Running gel -Tris – Hcl , pH 8.9
Anode
Tris – glycine pH 8.3 Buffer tank

41. Discontinuous polyacrylamide gel
electrophoresis –DISC-PAGE - 2
Sample application and run –
(-)
After injecting the sample, the
power is turned on and voltage is
Large molecules
adjusted to 60 mA. Small molecules
All -vely charged species start
migrating towards anode(+).
When marker dye reaches the top
of the gel, the power is stopped .
The separated zones in gel slab
are denatured by acid treatment Glycinate ions
(50% TCA) or (7-!) % acetic acid). Bromophenol blue
For nucleic acids, ethidium
bromide is used for staining.
Cl ions
For proteins, coomasie blue is (+)
used for staining.

42. Applications of electrophoresis
A versatile analytical tool
A valuable diagnostic A major tool in the
in forensics, molecular
tool in clinical pathology- analysis of human DNA
biology, genetics,
analysis proteins in body in Human Genome
microbiology and
fluids, Project.
biochemistry.
A separation tool in
A common laboratory serum proteins,
tool in the analysis of isoenzymes,
proteins, DNAs and immunoglobulin,
RNAs. abnormal haemoglobins
and serum lipoproteins.

43.  Dr.B.Victor is a highly experienced professor,
recently retired from the reputed educational
institution- St. Xavier‟ s College, Palayamkottai,
India-627001.
 He was the dean of sciences, IQAC coordinator and
assistant controller of examinations.
 He has more than 32 years of teaching and research
experience
 He has taught a diversity of college courses and
guided 12 Ph.D scholars.
 Send your comments to :
bonfiliusvictor@gmail.com