Electrophoresis is an electrokinetic process which separates charged particles in a fluid using a field of electrical charge. It is most often used in life sciences to separate protein molecules or DNA and can be achieved through several different procedures depending on the type and size of the molecules. The procedures differ in some ways but all need a source for the electrical charge, a support medium and a buffer solution. Electrophoresis is used in laboratories for the separation of molecules based on size, density and purity.An electric field is applied to molecules and as they are electrically charged themselves it results in a force acting upon them. The greater the charge of the molecule the greater the force applied by the electrical field and therefore the further through the support medium the molecule will move relative to its mass. Some example applications of electrophoresis include DNA and RNA analysis as well as protein electrophoresis which is a medical procedure used to analyse and separate the molecules found in a fluid sample (most commonly blood and urine samples).Different types of gels are usually used as the support medium for electrophoresis and this may be in slab or tube form depending on which is more beneficial. Gel slabs enable many samples to be run simultaneously and so are frequently used in laboratories. However, tube gels give a better resolution of the results so are often chosen for protein electrophoresis. Agarose gel is commonly used for electrophoresis of DNA. It has a large pore structure allowing larger molecules to move easily but it is not suitable for sequencing smaller molecules. Polyacrylamide gel electrophoresis (PAGE) has a clearer resolution than agarose gel making it more suitable for quantitative analysis. This makes it possible to identify how proteins bind to DNA. It can also be used to develop an understanding of how bacteria is becoming resistant to antibiotics through plasmid analysis.

1. ELECTROPHORESIS
S A K S H I N A Y A K
M S C . 1 S T S E M
I N S T. O F B I O M E D I C A L S C I E N C E S

2. INTRODUCTION
It is a physical method of analysis which involves separation of the
compounds that are capable of acquiring electric charge in conducting
electrodes.
It may be defined as the migration of the charged particle through a
solution under the influence of an external electrical field.
Ions that are suspended between two electrodes tends to travel towards
the electrodes that bears opposite charges.
It was discovered by Tselius in 1930s.

3. ELECTROPHORETIC MOBILITY
• The velocity (v) of charged molecule in an electric field:
v = Eq / F
where ,
• F = frictional coefficient, which depends upon the mass and
shape of the molecule.
• E = electric field (V/ cm)
• q = the net charge on molecule
• v = velocity of the molecule.

4. FACTORS AFFECTING ELECTROPHORETIC
MOBILITY
• Charge – Higher the charge greater the electrophoretic mobility.
• Size – Bigger the molecule greater are the frictional and electrostatic
forces exerted on it by the medium. Consequently, larger particles
have smaller electrophoretic mobility compared to smaller particles.
• Shape – Rounded contours elicit lesser frictional and electrostatic
retardation compared to sharp contours. Therefore globular protein
move faster than fibrous protein.

5. TYPES OF ELECTROPHORESIS
• Moving boundary electrophoresis
• Zone electrophoresis:
a) Paper Electrophoresis
b) Gel Electrophoresis

6. MOVING BOUNDARY ELECTROPHORESIS
• It was first developed by A.Tiselius in the 1930s.
• The moving boundary method allows the charged species to
migrate in a free moving solution without the supporting
medium.
• INSTRUMENTATION:
• Consists of a U shaped glass cell of rectangular cross
section, with electrodes placed on the one each of the
limbs of the cell.
• Sample solution is introduced at the bottom or through
the side arm, and the apparatus is placed in a constant
temp. bath at 40* C.

7. ZONE ELECTROPHORESIS
• It involves the migration of the charged particle on
the supporting media.
• Discrete zones are formed .
• Paper, Cellulose acetate membrane, Starch Gel, Poly
acrylamide.
• Components separated are distributed into discrete
zone on the support media.
• Supporting media is saturated with buffer solution,
small volume of the sample is applied as narrow band.

8. ZONE ELECTROPHORESIS

9. TECHNIQUES OF ZONE
ELECTROPHORESIS
• Paper electrophoresis
• Gel electrophoresis :
a) Agarose gel (AGE)
b) Polyacrylamide gel(PAGE)

10. GENERAL METHOD OF OPERATION
• Saturation of the medium with the buffer
• Sample application.
• Electrophoretic separation.
• Removal of the supporting media
INSTRUMENTATION:
• Electrophoretic chamber
• Electrodes
• Supporting/ Stabilizing media. (inert to sample and to any developing reagents)

11. ELECTROPHORESIS EQUIPMENT

12. PAPER ELECTROPHORESIS
• This technique is usefull for the separation of the small charged molecules such as
amino acids and small proteins.
• A strip of filter paper is moistened with buffer and the ends of the paper are
immersed in the buffer reservoir containing the electrodes.

13. PAPER ELECTROPHORESIS

14. PAPER ELECTROPHORESIS
• FILTER PAPER : It is the stabilizing medium. We can use whatman filter paper, cellulose acetate
filter paper or chromatography paper.
• APPARATUS : Power pack, electrophoretic cell that contains electrodes, buffer reservoirs, support
for paper, transparent insulating cover.
• SAMPLE APPLICATION : The sample may be applied as a spot(about 0.5 cm in diameter)or as a
uniform streak.
• ELECTROPHORETIC RUN: The current is switched on after the sample has been applied to the
paper and the paper has been equilibrated with the buffer. The types of buffer used depends upon
the type of separation. Once removed, the paper is dried in vaccum oven.
• DETECTION: To identify unknown components in the resolved mixture the electrophoretogram may
be compared with another electrophoretogram on which standard components have been
electrophoresed under identical conditions

15. GEL ELECTROPHORESIS
• It is a technique used for the separation of Deoxyribonucleic acid, Ribonucleic acid
or protein molecules according to their size and electrical charge using an electric
current applied to a gel matrix.
• Gel is a cross linked polymer whose composition and porosity is chosen based on
the specific weight and porosity of the target molecules.
• Types of Gel:
Agarose gel (AGE).
Polyacrylamide gel (PAGE).

16. GEL ELECTROPHORESIS
• Slab gel electrophoresis can have either a horizontal or vertical format .
• Sample is introduced into the wells at the top of the gel.

17. HORIZONTAL/SUBMARIN-E GEL
ELECTROPHORESIS
VERTICAL GEL ELECTROPHORESIS

18. CASTING OF THE GEL

19. GEL CASTING TRAYS
• Available in a variety of sizes and composed of
UV-transparent plastic.
• The open ends of the trays are closed with
tape while the gel is being cast, then removed
prior to electrophoresis.

20. LOADING OF THE GEL
• Carefully place the pipette tip over a well and gently expel the sample .The
sample should sink into the well. Be careful not to puncture the gel with the
pipette tip.

21. RUNNING OF THE GEL

22. STAINING

24. AGAROSE GEL
• It is a disaccharide consisting of galactose and 3,4 - anhydrogalactose.
• A highly purified uncharged polysaccharide derived from agar.
• Used to separate macromolecules such as nucleic acids, large proteins and protein
complexes.
• It is prepared by dissolving 0.5% agarose in boiling water and allowing it to cool
to 40°C.

25. AGAROSE GEL
• ADVANATGES:
• Easy to prepare and small concentration of agar is required.
• Resolution is superior to that of filter paper.
• Large quantities of proteins can be separated and recovered.
• Sharp zones are obtained due to less adsorption.
• Recovery of protein is good, good method for preparative purpose

26. POLYACRYLAMIDE GEL
• It is prepared by polymerizing acryl amide monomers in the
presence of methylene-bis-acrylamide to cross link the monomers.
• Structure of acrylamide (CH2=CH-CO-NH2 ).
• Polyacrylamide gel structure held together by covalent cross-
links.
• Polyacrylamide gels are tougher than agarose gels.
• It is thermostable, transparent, strong and relatively chemically
inert.
• Gels are uncharged and are prepared in a variety of pore sizes.
• Proteins are separated on the basis of charge to mass ratio and
molecular size, a phenomenon called Molecular sieving

27. PAGE
ADVANTAGES:
• Gels are stable over wide range of pH and temperature.
• Gels of different pore size can be formed.
• Simple and separation speed is good comparatively.
Types :
a) Native page
b) Denatured or SDS page

28. NATIVE PAGE(ISOELECTROPHORESIS)
• No denaturing agents.
• Proteins separated based on size, charge and shape .
• It is used when we want to keep the protein active to study conformation , binding
of other proteins .
• VISUALIZATION :
• Ethidium bromide .
• Silver or coomasive blue dye .

29. NON-NATIVE (SDS PAGE)
• It is negatively charged detergent (sodium dodecylsulfate).
• It is used to denature and linearize the proteins .
• It coated the proteins with negative charge.
• It is useful for monitoring protein purification as separation based on the size of the
particles.

30. STAINING OF DNA
• To make DNA fragments visible after electrophoresis
, the DNA must be stained .
• The favorite : Ethidium bromide .
• When bound to DNA it fluoresces under UV
light(reddish –orange color).
• Sensitive – detects 0.1ug of DNA.
• Other alternatives for ethidium bromide :
 Syber safe
 Xylene cyanol

31. 2D- GEL ELECTROPHORESIS
• In the first dimension, proteins are
resolved in according to their isoelectric
points (PI) using immobilized pH
gradient electrophoresis (IPGE),
isoelectric focusing (IEF), or non-
equilibrium pH gradient electrophoresis.
(Horizontal separation).
• In the second dimension, proteins are
separated according to their
approximate molecular weight using
SDS-PAGE. (Vertical separation).

33. CAPILLARY ELECTROPHORESIS
• It is the technique of performing electrophoresis in buffer filled, narrow-bore capillaries, normally
from 25 to 100 mm in internal diameter (ID).
• A high voltage (typically 10-30 kV) is applied.
• Capillaries are typically of 50 μm inner diameter and 0.5 to 1 m in length.
• Due to electroosmotic flow, all sample components migrate towards the negative electrode.
• The capillary can also be filled with a gel, which eliminates the electroosmotic flow. Separation is
accomplished as in conventional gel electrophoresis but the capillary allows higher resolution, greater
sensitivity, and online detection.
• The capillary is filled with electrolyte solution which conducts current through the inside of the
capillary. The ends of the capillary are dipped into reservoirs filled with the electrolyte.
• Electrodes (platinum) are inserted into the electrolyte reservoirs to complete the electrical circuit.

34. INSTRUMENTATION
• A buffer filled fused silica
capillary.
• Two electrode
• High voltage supply (5-30Kv)
• Sample injector
• Detector
• Buffer solutions

35. CAPILLARY ELECTROPHORESIS
• Detectors :
• Detectors similar to those used in GC, HPLC
can be used.
• Majority of instruments have UV- visible
detectors.
• The mass spectrophotometers are frequently
used to give structural information .
• Alternative detectors modes include
fluorescence , laser induced fluorescence.

36. CAPILLARY ELECTROPHORESIS
• ADVANATGES :
• Easy and predictable
selectivity.
• Small sample required
• Fast separation.
• Easily coupled to MS
• DISADVANTAGES :
• Cannot do preparative scale
separations.

37. PULSED FIELD GEL ELECTROPHORESIS
• It is a technique used for the separation of large molecules of DNA by
applying to a gel matrix in an electric filed that periodically changes
direction .
• The voltage is periodically switched among three directions; one that
runs through the central axis of the gel and two that run at an angle
of 60 degrees either side. The pulse times are equal for each direction
resulting in a net forward migration of the DNA.
• This procedure takes longer than normal gel electrophoresis due to the
size of the fragments being resolved and the fact that the DNA does
not move in a straight line through the gel.

38. APPLICATIONS
• DNA Sequencing
• Medical Research
• Protein research/purification
• Agricultural testing
• Separation of organic acid, alkaloids, carbohydrates, amino acids, alcohols, phenols,
nucleic acids, insulin.
• In food industry
• It is employed in biochemical and clinical fields i.e. in the study of protein mixtures
such as blood serum, hemoglobin and in the study of antigen- antibody interactions.
• Electrophoresis in combination with autoradiography is used to study the binding of
iron to serum proteins.
• Used for analysis of terpenoids , steroids and antibiotics.
• For testing purity of thyroid hormones by zone electrophoresis.
• Paper chromato-electrophoresis is used to separate free Insulin from plasma proteins.
• It is used for diagnosis of various diseases of kidney , liver and CVS.
• It is also used for separation of Scopolamine and Ephedrine using buffer at PH 4.2.
• Electrophoresis is also used for separation of carbohydrates and vitamins.

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