This document provides an overview of electrophoresis, including definitions, principles, types, and applications. It discusses four main types of electrophoresis: paper electrophoresis, gel electrophoresis, capillary electrophoresis, and iso-electric electrophoresis. Paper and gel electrophoresis are described as zone electrophoresis techniques that separate components into bands on a supporting medium like paper or gel. Capillary electrophoresis allows separation in free solution within a thin capillary tube. Iso-electric electrophoresis separates molecules based on their iso-electric point, the pH where they have no net charge. The document outlines the principles, instrumentation, and applications of each electrophoresis technique.
3. ELECTROPHORESIS:
DEFINITION:
Electrophoresis may be define as migration of charged particle
through solution under influence of external electrical field.
Ions that are suspended between two electrodes tends to travel towards
that electrodes that bears opposite charges.
4. PRINCIPLE:
The migration velocity ‘V’ of ions in electric field is equal to product of
field strength ‘E’ and electrophoretic mobility µ.
V = Eµ
Electrophoretic mobility ∝ Ionic charges
Frictional retarding factor
If two species differ in charge or either frictional force they get easily
separated from each other but neutral species are not separated.
5. FACTORS AFFECTING:
1) ELECTROLYSIS: Electrophoresis is accompanied by electrolysis which
leads to formation of microscopic bubbles. These bubbles get accumulated
on electrodes, as a result electrical contact of electrode with buffer solution
is lost and electrode resistance increase gradually, which leads to migrate
particle at irregular rate and affect separation process.
2) IONIC STRENGTH: In electrophoresis,
Electrophoretic mobility ∝ 1
√ Ionic Strength.
3) pH and Other Characteristic: The electrophoretic mobility is greatly
affected by pH of buffer particularly affected when sample is either weak
acid or weak base.
pH of buffer ∝ Ionic strength.
6. 4) ELECTRO-OSMOSIS: In this technique there often flow of water
under influence of voltage gradient which is known as electro-
osmosis. The rate of osmosis is influenced by conc. of ionic
in water.
5) HEAT: Heat causes production of convection current which
disturb the electrophoretic pattern.
7. TYPES OF ELECTROPHORESIS:
1) ZONE ELECTROPHORESIS:
Paper Electrophoresis
Gel Electrophoresis
2) MOVING BOUNDARY ELECTROPHORESIS:
Capillary Electrophoresis
Iso-electrophoresis
8. ZONE ELECTROPHORESIS:
Any electrophoretic technique in which components separated into zones or bands in a buff
er, and stabilized in solid, porous, or any other support medium –
e.g. filter paper, agar gel, or polyacrylamide gel.
Zone electrophoresis is an electrophoretic separation technique typically used for analyzing
proteins, nucleic acids, and biopolymers.
During the process, different species in a sample are transported in a continuous
electrolyte buffer system, subject to a potential gradient.
9. This is most prevalent electrophoretic technique of these day.
In these type of electrophoresis separation process is carried out on stabilized
media.
They produces zones of protein that are heterogeneous and separated from each
other.
An inert polymeric support media is used between the electrode to separate and
analyze the sample.
Supporting media such as Adsorbent paper, gel or starch etc.
Classified according to type and structure of support material e.g. : PAGE,
AGE, CAE etc.
Media minimizes the mixing of sample.
Analysis and purification of molecule from gel is much easier than moving
boundary electrophoresis.
10.
11. PAPER ELECTROPHORESIS:
Paper electrophoresis comes under zone electrophoresis.
PRINCIPLE:
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 protein
which can have either a net positive or net negative charge.
12. EQUIPMENTS:
Equipment required for the paper electrophoresis basically consist of two
parts:
1) POWER PACK: Power pack provides a stabilized direct current and
has control for both voltage (0 to 500v) and current output (0 to 150mA).
2) ELECTROPHORETIC CELL: It contain electrode, buffer
reservoirs ,a support for paper and transparent insulating cover. The
electrode are usually made of platinum.
13.
14. WORKING:
A Long strip of filter paper is moistened with suitable buffer solution of
desired pH and sample is transversely across the central part of strip.
End are fixed to dip in buffer solution in two containers fitted with
electrodes.
The charged particles of sample migrate along the strip toward electrodes
of opposite polarity.
Homogeneous group of particle migrate as a separate band.
Electrophoresis is out for 16 – 18 hours.
Proteins are stain to make them visible( bromophenol blue).
15. TYPES OF PAPER USED:
i. Whatmann paper no. 1,2,3 etc.
ii. Eaton- Dikemann 301-85, 320 and 352.
iii. Munketells 20/50.
iv. Large sheet of Whatmann thin paper 1 for large volume sample.
TYPES OF PAPER ELECTROPHORESIS:
i. Horizontal paper electrophoresis.
ii. Vertical paper electrophoresis.
iii. Continuous paper electrophoresis.
16. Horizontal paper electrophoresis in which strip is positioned in apparatus
without any distortion and lower part is immersed in solution.
Paper is allowed to stand for 1 hour.
Separation is achieved in 12 to 14 hours.
17. In vertical mode the separation and migration of ions is assisted by gravity in
addition to electrophoretic mobility.
18. In this method the thin sheet of filter paper is usually used as supporting
medium.
It is used for preparative sample
19. APPLICATION:
Paper electrophoresis has emerged as a simple, inexpensive and accurate lab procedure
for various research and clinical studies.
Clinical Application of paper electrophoresis include study of sickle cell disease,
haemoglobin abnormalities, separation of blood clotting factor and serum plasma
protein from blood sample.
20. It has also been used in separation and identification of alkaloids.
Paper electrophoresis also be used for testing water sample, toxicity of water
and other environmental components.
Drug testing industries uses PE to determine presence of illegal drugs crime
suspects.
21. GEL ELECTROPHORESIS:
Gel electrophoresis is process for separation and analysis of macromolecules
like RNA, DNA, Protein and their fragments based on their size and charges.
Gel electrophoresis uses gel as an anti-convective medium and/or sieving
medium during electrophoresis.
Gel suppress the thermal convection caused by electric field. Gel can simply
serve to maintain the finished separation so that the post electrophoresis
strain can be applied.
By placing the substance to be separated in wells of the gel and applying an
electric current, allows the molecule to move through matrix at different
rates towards the anode and cathode .
As they move through the gel the larger particle will held up as they try to
pass through the pores of gel while the small particle will move at faster rate.
Hence it allows the separation based on size and charges present on particle.
22. INSTRUMENT AND REAGENT:
1) ELECTROPHORESIS APPRATUS
2) POWER SUPPLY
3) BUFFER
4) SUPPORTING MEDIA
5) DETECTION
6) QUANTIFICATION.
23.
24. ELECTROPHORETIC APPARATUS:
The casting tray is made up of plastic or glass.
The comb contain varying number of teeth which is used for formation of well in
proper manner desire for separation.
25. BUFFER SOLUTION:
Buffers in gel electrophoresis are used to provide ions that carry current and
maintain pH at relative constant value.
Buffers usually used are EDTA/TAE.
26. POWER SUPPLY:
Electrodes are connected to their respective terminal of electrophoresis chamber
and to power supply with control rate of current flow.
The best resolution of fragment larger than about 2 kb is attained by applying
not more than 5V/cm to the gel.
27. SUPPORTING MEDIA (GEL) :
1) Starch
2) Agarose
3) Cellulose acetate
4) Polyacrylamide gel
The kind of separating matrix depend on the type of molecule to be
separated.
Agarose and polyacrylamide gels are crossed linked and forms sponge like
structure.
It is important that supporting media should be electrically neutral as
presence of charge may cause molecule migration retardation.
Agarose gel have large pore size and used for separating larger DNA
molecule and polyacrylamide gel is used to obtain high resolution separation
for smaller DNA molecule.
28.
29.
30. APPLICATION:
Application of gel electrophoresis in estimation of size of DNA molecules and
investigation of DNA cleavage efficiency of small molecules, for example, are
extensively used in molecular biology.
Gel electrophoresis is also commonly used in plant breeding and genomics
for genotyping with molecular markers.
Todays, gel electrophoresis has been used in analysis of PCR
products especially in molecular genetic diagnosis or genetic fingerprinting
with the measurement and analysis are mostly done with specialized software.
31. MOVING BOUNDARY
ELECTROPHORESIS:
PRINCIPLE:
The moving boundary allows the charged species to migrate in free
moving solution without supporting medium.
INSTRUMENTATION:
Consist of U shaped glassed rectangular cross section tube with
electrode placed on the each terminal.
Sample solution is introduced to bottom through side arm.
Apparatus is placed in constant temperature bath at 40C
Detection is done by measuring refractive index throughout the
solution.
32.
33. While there is specialized application for moving boundary
electrophoresis ( capillary electrophoresis) it is not common
technique for two reasons.
1) The resolution between different population of molecule can be
lost due to fluid motion if special precaution are not taken . Fluid
motion can occur through physical distribution or through thermal
convection .
2) The time and electrophoretic distance needed to separate
molecules with similar charges and masses may be impractical.
34. CAPPILARY ELECTROPHORESIS:
Capillary electrophoresis is an analytical technique that separates ions based
on their electrophoretic mobility with applied voltage.
Separation method carried out in a buffer-filled capillary tube that is typically
10 to 100 μm in internal diameter and 40 to 100 cm in length.
The tube extends between two buffer reservoirs that also hold Pt electrodes.
The sample is introduced into one end of the tubing, and a dc potential in
the 10 to 30 kV range is applied between the two electrodes throughout the
separation.
The separated analyzed are observed by a detector at the end of the capillary
opposite the end where the sample was introduced.
35. Instrumentation:
Electrophoretic separations use two platforms:
slab electrophoresis and capillary electrophoresis.
The basic instrumentation for capillary electrophoresis is shown here and
includes a power supply for applying the electric field, anode and cathode
compartments containing reservoirs of the buffer solution, a sample vial
containing the sample, the capillary tube, and a detector. The sample and the
source reservoir are switched when making injections.
36. Capillary tubes are made from fused silica coated with a 15–35 μm layer of
polyimide to give it mechanical strength.
The inner diameter is typically 25–75 μm—smaller than the internal diameter of a
capillary GC column—with an outer diameter of 200–375 μm.
The capillary column’s narrow opening and the thickness of its walls are important.
When an electric field is applied to the buffer solution within the capillary, current
flows through the capillary.
37.
38. APPLICATION:
The use of capillary electrophoresis (CE) in genetic analysis applications has
largely replaced the use of gel separation techniques due to significant gains
in workflow speed, throughput, and ease of use.
Forensic labs across the world utilize capillary electrophoresis to analyze a
wide range of evidence collected from crime scenes. One of its first uses was
for the analysis of explosive compounds and gunshot residues. Capillary
electrophoresis is considered to be a fast, sensitive, and reliable detection
method of explosives that can work with a high workload.
Capillary electrophoresis has been implemented in clinical research to
analyze peptides and proteins, chiral pharmaceuticals, and other small
molecule drugs. Studies have shown the technique’s use in assessing purity
and structural formation.
39. Capillary electrophoresis is used for multiple diseases that require enhanced
diagnostic methods, with rapid methods of diagnosis emerging for
microalbuminuria, Alzheimer’s and Parkinson’s diseases, neuroblastoma, and
urinary stone disease using the technique.
A range of capillary electrophoresis is used by the industrial sector to
determine various analytes in a wide range of sample matrices. Capillary
electrophoresis in industry is used to analyze products such as; food additives,
herbicide, animal nutrition, and detergents.
40. Iso-electric focusing:
Iso-electric focusing began in 1964.
This technique was popularized by H. svensson in Sweden.
Iso-electric focusing is technique for separating different amphoteric molecule by
based on their iso-electric point.
Iso-electric point is pH at which net charge on protein becomes ZERO.
ISOELECTRIC
ELECTRIC- charge
ISO- Same
41. IEF is performed in pH gradient.
Protein are amphoteric molecule with acidic and basic buffering groups.
1) In basic environment the acidic group become negatively charged.
2) In acidic environment the basic group become positively charged.
3) Protein with same molecular weight will be separate out by pH.
Proteins are positively charged in solution at pH value below pI and
migrate toward cathode.
Protein are negatively charged in solution at pH value above pI and migrate
towards anode.
42.
43. Protein stop migrating when pH= pI .
Stained protein are easy to visualize.
Highly stable ampholytes are molecule with specific pKa to give specific
and unchanging pH gradient.
Separation is obtain by applying a potential difference across gel that
contain pH gradient.
They required solid support such as agarose gel and polyacrylamide gel.
They contain synthetic buffer called ampholyte that smooth the pH
gradient.
Ampholyte= Mixture of polyamino-polycarboxylic acid
44. APPLICATION:
IEF is highly sensitive analytical technique and is practically useful for
studying micro-heterogeneity in protein.
EXAMPLE: Protein may show single band on SDS gel but may show 3 to
4 bands on IEF gel.
They are used to separate isoenzyme.
2D gel electrophoresis is application of IEF , here protein is first separated
based on the pI and then based on molecular weight using PAGE.
Widely used for separation and identification of serum protein.
The greatest advantage is its high resolution resulting in greater separation.
