Sie sind auf Seite 1von 63


Husniah Batool
M.Phil Immunology(part-1)
University of Health Sciences, Lahore
 Electrophoresis is a technique whereby
charged molecules in solution, chiefly
proteins and nucleic acids, migrate in
response to an electrical field.

 It is widely used analytical technique for

separation of biological molecules i.e
plasma protein, lipoproteins,
immunoglobulins , abnormal heamoglobin
 In 1937, a Swedish scientist Arne Tiselius
developed an apparatus for measuring the
movement of protein molecules, called
Moving Boundary apparatus.
 In 1940, zone electrophoresis was
introduced, which uses a solid medium
(e.g., gel) and allows staining for better
resolution or visualization of the
separation of molecules.
 Then in 1960, the capillary
electrophoresis was developed to provide
a versatile electrophoresis technique.
This type of electrophoresis allows
separation of molecules using aqueous
and solid mediums.
 Electrophoresis is a physical method of
analysis which involves separation of the
compounds that are capable of acquiring
electric charge in conducting electrodes.
 When charged
molecules are
placed in an
electric field, they
migrate toward
either the positive
(anode) or
negative (cathode)
pole according to
their charge.
 Serum proteins are negatively charged at pH
8.6 (a buffer maintains a constant pH) and
they move towards the anode at the rate
dependent on their net charge.

 Bio molecules move with a speed dependent

on their charge, shape, size and separation
occures on the basis of molecular size.
 Two basic requirements
1. There must be a difference in how analytes
will interact with the separation system so
analytes must have different migration
time and migration distance.
2. The bands or peaks for the analytes must
be sufficiently narrow to allow them to be
 To determine the number, amount and mobility
of components in a given sample or to separate

 Determination of molecular weight of proteins

and DNA sequencing.

 To obtain information about the electrical

double layers surrounding the particles.
 Factors related to sample
 Higher the charge greater electrophoretic
 Bigger molecules……greater friction and
electrostatic force exerted on it by medium.
 Round contours…. Lesser frictional and
electrostatic retardation compared to sharp
contours. So globular proteins move faster than
fibrous proteins
 Support medium
 Carries applied electric current.
 Establishes pH at which electrophoresis takes
 Determines electrical charge on solute.

 Provide separation through molecular
 Record of results.
 Environmental factors
 pH
 Proteins are amphoteric substances.
 Each protein has its own characteristic
charge properties depending on the number
and kinds of amino acids carrying amino or
carboxyl groups
 Denaturation of proteins
 Properties of electric field
 Applied voltage.
 Mobility = (Applied voltage)(net charge)
Friction of molecule
 Current….V=IR
 Resistance….will determine the amount of
heat generated during electrophoresis.
 Isclassified on the basis of presence and
absence of solid support medium or matrix
through which charged particle move.
 Zone Electrophoresis
a. Paper Electrophoresis
b. Gel Electrophoresis
c. Thin Layer Electrophoresis
d) Cellulose acetate Electrophoresis
 Moving Boundary Electrophoresis
a. Capillary Electrophoresis
b. Isotachophoresis
c. Isoelectric Focussing
d. Immunoelectrophoresis
 Migration of charged molecules
 Support medium
 Porous i.e agarose
 Can be dried and kept
 Same pH and field strength throughout.
 Separation based upon electrophoretic
 Separates macromolecules colloides i.e
protein in serum, urine, CSF, erythrocytes,
nucleic acid.
 Commonly used for resolution and analysis of
small particles.
 Not suitable for larger particles(proteins),
because adsorption and surface tension
denatures macromolecules causing poor
 Two different methods are used.
 Dry application procedure, sample of solutes
dissolved in distilled water or volatile buffer
is applied as a small spot “origin line”.
 Appropriate standards of known compound
are applied on other location on origin line.
 Solvent containing samples
evaporated…paper is dampened with
electrophoresis buffer by uniform spraying or
 Advantage: small initial sample spots
and better resolution of similarly mobile
 Disadvantage: Dipping and spraying
requires considerable skills to avoid
spreading of applied sample.
 Sample dissolved as concentrated solution in
distilled water are applied on paper
predampened on electrophoresis buffer.

 Placed in electrophoresis chamber and

electric field is applied….. resistance to
current flow… generation of heat….. drying
paper……….. further increase in resistance…..
change in current flow… distort the
migration of molecules.
 Modern paper electrophoresis system have
been designed.
 Cooled flat-bed system to dissipate the heat
or operate in a cooled bath of inert and
nonpolar solvent (e.g. Varsol, a petroleum
 This solvent absorbs the heat generated by
the system without mixing with the water,
buffers, or samples on the papers.
 Gelelectrophoresis is performed by applying
a sample to a gel support that is then placed
into an electric field.

 Severalsamples are applied to the gel and

allowed to migrate along the length of the
support in the presence of an applied
 The separation is stopped before analytes
have left the end of gel, with the location
and intensities then being determined.
 Buffer
solution is used to conduct electricity
through whole setup of gel electrophoresis.

 Moleculeswill migrate through gel depending

upon their size and shape.

 Velocity is related to distance traveled.

 Greater distance traveled…. Greater
 Electrophoretic mobility
 Support contains a running buffer with
ions that carry a current through the
support when an electric field is applied.

 Once samples have been placed on the

support, the electrodes are connected to
a power supply and used to apply a
voltage across the support.
 This electric field is passed through the
system for a given amount of time, causing
the sample components to migrate.

 After the electric field has been turned off,

the gel is removed and examined to locate
the analyte bands.
 The samples in gel electrophoresis are
applied to small “wells” that are made in the
gel during its preparation.

A sample volume of 10–100 μL is then placed

into one of these wells by using a
 Direct detection
 Visually…. intensely colored proteins like
 Scanning device…. Densitometer
 Stains or reagents….
 For protein… Amido black, Coomassie
Brilliant Blue.
 Low conc. Proteins...... Silver nitrate
 DNA bands…. Etlhidium bromide
 “BLOTTING” Technique
 Transfer a portion of the analyte bands to a
second support ( nitrocelluose), where they
are reacted with a labelled agent.
 Southern blot: detect specific sequences of
 Northern blot: detect specific sequence of
RNA by labeled DNA probe.
 Western blot: detect specific protein.
 Types of gel commonly used for gel
 Agarose
 Polyacrylamide
 Starch
 One dimensional
2. Native: Non denaturing PAGE, also called
native PAGE, separates proteins according to
their mass:charge ratio.
 For preparation of purified and active protein
3. IEP
 Two dimensional.
 Two-dimensional PAGE (2D-PAGE) separates
proteins by isoelectric point in the first
dimension and by mass by SDS-PAGE in the
second dimension.
 Agarose is a linear polymer extracted from
seewead that forms a gel matrix by hydrogen

 Used to separate fragments of DNA and RNA

in the matrix of agarose.

 DNA has negative charge and migrates

towards positive electrode.
 Density
and porosity of gel matrix is
determine by concentration of agarose used.

 Greater agarose concentration, smaller pore

size in gel matrix and more difficulty for
larger pieces of DNA to move through.
 Polyacrylamidegels are used to separate
protein molecules based on size, shape and

 Polyacrylamideis specifically used for

proteins because it provides environment
where proteins will not become denatured.

 Allowingdifferent size proteins to move at

different rate.
 Sinceproteins have different size, shapes
and complex structure (secondary, tertiary
and quaternary).

 To have proteins with linear structures

Sodium dodacyl sulphate (SDS) is used.

 SDSis an anionic detergent that can dissolve

hydrophobic molecules resulting in proteins
with linear structures.
 Estimation of size of DNA and protein

 Analysis
of PCR products i.e in molecular
genetic diagnosis and genetic fingerprinting.

 Separation of restricted genomic DNA and

 This method allows charged species to
migrate in a free moving solution.
 Samples are placed in a U shaped tube that
has been filled with a buffer.
 Electric field is applied at the ends of tube.
 Separation takes place on the basis of
difference in mobilities.
 Bands are obtained which are located by
optical system.
 Materialto be analyzed and the
electrophoresis medium (a conducting liquid,
usually aqueous) are placed in a long, fine-
bore capillary tube 50-100cm long and 25-
100 µm inside diameter.
 Small sample is placed at one end of
capillary and 20-30kV voltage is applied.
 Analytes are separated and detected at the
other end of the capillary.
 Advantages: high resolution speed and high
sensitivity for extremely small samples.
 Separation of DNA molecules which differ in
single nucleotide.
 Separation of uncharged molecules by
including charged micelles of detergents.
 Separates proteins according to the iso-
electric points.
 Point of zero net charge is called isoelectric
 Is ideal for separation of amphoteric
 Generally protein readily crystallise at
isoelectric point.
 Most proteins have isoelectric point 5-9
 Separation is achieved by applying a
potential difference across a gel that
contains a pH gradient.
pI = pH at which the
protein has equal
amount of positive
and negative charges
(the net charge is
if pH < pI = +ve
charge and vice
 Migration of small ions.
 Discontinuous electrolyte system
 Leading electrolyte (L ions) i.e chloride
 Trailing electrolyte (T ions) i.e glycinate
 Apply sample solution at interphase of L & T
 Apply electric field…… each type of ions
arrange between L & T ions…. Discrete zones
will form.
 Separates small ions, cations, amino acid
peptides, nucleotides, nucleosides and
 Also called gamma globulin electrophoresis,
or immunoglobulin electrophoresis, is a
method of determining the blood levels of
three major immunoglobulins, IgM, IgG, and
 Immunoelectrophoresis is a powerful analytical
technique with high resolving power as it
combines separation of antigens by
electrophoresis with immunodiffusion against an

 It is usually requested when a different type of

electrophoresis, called a serum protein
electrophoresis has indicated a rise at the
immunoglobulin level.
 Serum is electrophoresed to separate out the main
protein fractions.

 Trough is cut in the gel parallel to the line of

separation. Antiserum is placed in the trough.

 Gel is incubated for 18 to 24 hours.

 Double diffusion occurs at right angles to the

electrophoretic separation.

 Precipitin arcs develop where specific antigen–

antibody combination takes place.
 These arcs can be compared in shape,
intensity, and location to that of a normal
control serum to detect abnormalities.
 Drugs that may cause increased
immunoglobulin levels include therapeutic
gamma globulin, hydralazine, isoniazid,
phenytoin (Dilantin), procainamide, oral
contraceptives, methadone, steroids,
and tetanus toxoid and antitoxin.

 This is mainly because prior immunizations

lead to the increased immunoglobulin levels
resulting in false positive results.
 Immunoelectrophoresis is not quantitative,
it is being replaced by a procedure called
immunofixation, which is more sensitive and
easier to interpret.
 Protein electrophoresis separates the
globulins from albumin and resolves the
major proteins of serum into patterns
that may be highly specific for some
 Identification of these patterns is a useful
screening method to be followed by more
specific confirmatory procedures to
identify and quantitate aberrant protein
Evaluation of separated protein fractions

Serum proteins are separated

into 6 groups:
α1 - globulins
α2 - globulins
β1 - globulins
β2 - globulins
γ - globulins