1. Electrophoresis
Dr. Ajit Kumar Singh
PGT 1st year (Laboratory medicine)
Chittaranjan National Cancer Institute , Kolkata
Moderator
Dr. Garima Chauhan
MD Biochemistry
Assistant professor
Department of Laboratory medicine
Chittaranjan National Cancer Institute , Kolkata
2.
3. General principles of electrophoresis
• Electrophoresis is the migration of charged particles or molecules in a
medium under the influence of an applied electric field.
• A separation technique
• Simple, rapid , and highly sensitive
Used in clinical laboratories to separate charged molecules :
• Proteins in body fluids : serum ,urine , CSF
• Proteins in erythrocytes : Hemoglobin
• Nucleic acid : DNA , RNA
6. General principles of electrophoresis
Force experienced by particle in an electrical field is given by Coulomb’s law :
F = ZqE ………………………………………… equation (1)
The viscous resistance of the medium to the motion given by Stokes’ law :
F’ = 6πrηv …………………………………….. Equation (2)
The viscous resistance of the medium just balances the driving force :
F = F’
ZqE = 6πrηv
Electrophoretic mobility (U) = v/E = Zq/6πrη
7. Factors affecting electrophoresis
Inherent factors
Net charge of the molecules
Size and shape of the molecules
Molecular weight
External environment
Solution pH
Electric field
Solution viscosity
10. 2. Buffer
The buffer in electrophoresis has two fold purpose
Carry applied electrical current
They set the pH at which electrophoresis is carried
Tris acetate EDTA (TAE) and tris borate EDTA (TBE) (Most common used buffer)
Buffer pH value
• Phosphate buffer around 7.0
• Tris -Glycine buffer (TG) more than 8.5
• Tris -Citrate-EDTA buffer (TCE) around 7.0
• Tris -EDTA buffer (TE) around 8.0
• Tris -Maleic acid -EDTA buffer (TME) around 7.5
• Lithium Borate - buffer (LB) around 8.6
11. 2. Buffer
The buffer in electrophoresis has two fold purpose :
• Carry applied electrical current
• They set the pH at which electrophoresis is carried out.
Thus they determine :
• Type of charge on solute.
• Extent of ionization of solute
• Electrode towards which the solute will migrate.
• The buffer ionic strength will determine the thickness of the ionic
cloud.
12. 3. Support Media
The support medium provides the matrix in which protein separation
takes place.
Various types of support media have been used in electrophoresis
and range from pure buffer solutions in a capillary to insoluble gels or
membranes of cellulose acetate.
- Starch gel
- Cellulose acetate
- Agarose
- Polyacrylamide gel (PAG)
13. 4. Stain
Separation type Stain
Serum proteins Amido Black (Naphthol Blue Black)
Coomassie Brilliant Blue (brilliant blue G)
Coomassie Brilliant Blue (brilliant blue R)
Ponceau S
Isoenzymes Nitrotetrazolium Blue
Lipoprotein Fat Red 7B (Sudan Red 7B)
Oil Red O
Sudan Black B
DNA
fragments
Ethidium bromide
CSF proteins Silver nitrate
Depends on nature of the molecule to be detected
14. 5. Quantification
A densitometer is used .
Essential features of a densitometer include :
• The ability to scan gels 25 to 100 mm in length
• Variable wavelength control over the range of 400 to 700 nm
• An integrating device with both automatic and manual selection
• Automatic indexing
16. DNA is negatively charged.
When placed in an electrical field, DNA will migrate toward the positive pole
(anode).
An agarose gel is used to slow the movement of DNA and separate by size.
17. Horizontal gel electrophoresis
Agarose gel electrophoresis is a method to separate
DNA, or RNA molecules by size.
This is achieved by moving negatively charged nucleic acid
molecules through an agarose matrix with an electric field
(electrophoresis).
Shorter molecules move faster and migrate farther than
longer ones .
18. At any given PH, exist in a solution as electrically charged species
either as a cation (+) or anion(-).
Under the influence of an electric field these charged particles will
migrate either to cathode or anode, depending on the nature of their
net charged
Electrophoresis is the movement of molecules by an electric
current.
Nucleic acid moves from a negative to a positive pole.
20. Steps followed in gel electrophoresis
• Mix agarose and buffer
• Boil mixture in microwave
• Cool the mixture at 65 and pour in
to chamber , comb inserted in to
chamber to make well
• Gel solidified at room temperature ,
comb remove wells remain
• Load the prepared samples in to gel
• Separate fragments by
electrophoresis
• Stain separated molecules and
measure distance
21. Electrophoretic Separation
When electrophoresis is performed following steps are typical
Excess buffer is removed from the support surface by blotting , taking
care that bubbles are not present
5 to 7 µL of sample is applied using a comb or a plastic template and is
allowed to diffuse into the gel; it is then blotted to remove the excess
The gel is placed into the electrode chamber
Electrophoresis is performed at specified current, voltage, or power
The gel is fixed, rinsed, and then dried
The gel is stained and redried and
The gel is scanned in a densitometer
22. Types of electrophoresis
1.Zone electrophoresis
Discrete zone formed
Example –
• Paper electrophoresis
• Agarose gel electrophoresis
• Poly acrylamide gel electrophoresis (PAGE)
23. Types of electrophoresis
2.Moving Boundary Electrophoresis
Involves the migration of charged molecules in a free moving solution,
without the presence of a supporting medium
Example –
• Capillary electrophoresis
• Microchip electrophoresis
24. Types of electrophoresis
1.Zone Electrophoresis
a. Paper electrophoresis
b. Agarose gel electrophoresis
c. Poly Acrylamide Gel Electrophoresis (PAGE)
d. Pulsed-field gel electrophoresis (PFGE)
e. SDS-PAGE
f. 2D electrophoresis
g. Immuno-electrophoresis(Rocket Electrophoresis)
I . Difference Gel Electrophoresis(DIGE)
2.Moving Boundary Electrophoresis
a) Capillary Electrophoresis
b) Iso-tachophoresis
25. A. Paper electrophoresis
Used in clinical investigations of serum and other body fluid
Disadvantage :
• Adsorbs proteins
• Poor conductivity
• Background staining
• OH groups of cellulose bind with proteins and retard electrophoretic
movements causing trailing of bands and poor resolution
Advantage :
• It is economical , Easy to use , non-toxic , can be stored easily
27. Agarose gel
• A linear polysaccharide (made-up of repeat unit of agarobiose-
alternating unit of galactose and 3,6-anhydrogalactose).
• Used in conc as 1% and 3%.
• The gelling property are attributed to both inter- and intramolecular
hydrogen bonding.
• Pore size is controlled by the % of agarose used.
• Purity of the agarose is based on the number of sulphate conc, lower
the conc of sulphate higher is the purity of agarose.
• Acts as a sieve for separating DNA fragments; smaller fragments
travel faster than large fragments
28. Combine the agarose powder and buffer solution. Use a flask
that is several times larger than the volume of buffer.
Agarose
29. Melting the Agarose
).
Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
30. B. Agarose gel electrophoresis
Agarose gel - porous material derived from red seaweed .
Gel material acts as a "molecular sieve” and electrically neutral .
Concentration affects DNA migration , used in conc as 1% and 3%.
Low conc. = larger pores better resolution of larger DNA
fragments
High conc. = smaller pores better resolution of smaller DNA
fragments
31. B. Agarose gel electrophoresis
• Effective separation range of agarose gels of various compositions for
separation of nucleic acids -
• % Agarose (wt / vol) Effective Separation Range (base pairs)
• 0.8 700–9000
• 1.0 500–7000
• 1.2 400–5000
32. C. Poly acrylamide gel electrophoresis (PAGE)
• Used up to 3-30% concentration
• lower concentration for DNA separation and higher concentration for
protein separation
33. C. Poly Acrylamide Gel Electrophoresis (PAGE)
% Acrylamide in Resolving Gel Effective Separation Range (Da)
• 7.5 45,000–200,000
• 10 20,000–200,000
• 12 14,000–70,000
• 15 5,000–70,000
• 20 5,000–45,000
34. Application
• For determination of the molecular weight of DNA
• For DNA sequencing
• To study the purity of DNA
• To analyze recombinant DNA molecule
• Can also separate RNA molecule, and its molecular weight can also be
determined using calibration curve similar as in DNA
35. D. Pulsed-field gel electrophoresis (PFGE)
• This technique was developed by Shwartz and Cantor in 1984.
• Separation of DNA in agarose gel by altering the strength and
direction of the electrical field between electrodes.
• It is used to separate high molecular weight DNA of several mega
bases, even whole chromosomes.
36. Application of PFGE
• PFGE is accurate and results are reproducible with good efficiency, it is used in
several areas.
• Molecular studies of food-borne pathogenic organisms such as Salmonella, E. coli
O157:H7, Shigella, Listeria, Campylobacter, etc.
• The wine industry uses PFGE to monitor the genetic stability of organisms
involved in the fermentation processes.
• PFGE is the first step in cutting and separating large DNA fragments before
cloning vectors.
• It is very useful in mapping applications such as mapping specific disease loci,
identifying chromosome rearrangements, RFLP, and DNA fingerprinting.
• Detecting related strains in case of hospital outbreaks
37. E. SDS-PAGE
• 1st known as the Laemmli method after its inventor- U.K. Laemmli
Upper Stacking gel :
• It has larger pores with a pH of 6.8
Lower Separating gel:
• It has smaller pores with a pH of 8
38. Applications
• To determine the molecular weight of proteins.
• To fractionate protein subunits
• To assess the purity of protein samples (because of high resolving
power).
• To determine the isoelectric point(Pi) of a protein.
• To separate isoenzymes.
• To fractionate proteins with higher resolution.
• To study mono, di and tri substituted derivatives of protein.
• To separate all amphoteric substances.
39. F. 2D electrophoresis
• The technique of IEF and SDS PAGE combined for fine separation of
polypeptides having only minute differences in pI and molecular
weight
• First separation by IEF
• Next separation according to mol wt(SDS PAGE) which separates
protein according to size at right angles to the direction of 1st
separation.
• Series of spots formed in gel which can be quantified, images
matched and compared with corresponding spots in related gels
40. G. Immuno-electrophoresis
(Rocket Electrophoresis)
Principle
• It combines the sensitivity of gel electrophoresis with the specificity of immune
reaction. This technique is used to determine the quantity of a given antigen.
Separation
• Molten agar solution, saturated with a suitable antibody that is complementary
against the test antigen to be quantified, is layered on a horizontal plate
• On the gel, wells are drilled where antigens are filled
• At the alkaline pH, Ag acquires a negative charge and migrates towards the anode
and interact with Ab to form Ag-Ab complex, immunoprecipitates
• The gel is then stained with CBB, immunoprecipitates will appear in the form of
rocket-shaped arcs.
41. • The test helps in identification and approximate quantization of
• various antigens in immunology.
• suspected monoclonal and polyclonal gammopathies.
• Used to analyze complex protein mixtures containing different
• antigens.
• The medical diagnostic use is of value where certain proteins are
• suspected of being absent (e.g., hypogammaglobulinemia) or
• overproduced (e.g., multiple myeloma).
• This method is useful to monitor antigen and antigen-antibody
• purity and to identify a single antigen in a mixture of antigens.
42. H. Difference Gel Electrophoresis(DIGE)
• Up to 3 different protein samples can be labeled with size and charge
matched fluorescent dyes (for example Cy3, Cy5, Cy2)
• the three samples are mixed, loaded and 2D electrophoresis is carried
out after which the gel is scanned with the excitation wavelength of
each dye one after the other, so we are able to see each sample
separately.
• This technique is used to see changes in protein abundance (for
example, between a sample of a healthy person and a sample of a
person with disease), post-translational modifications, truncations,
and any modification that might change the size or isoelectric point of
proteins.
43. Difference Gel Electrophoresis(DIGE)
• Since the proteins from the different sample types (e.g.
healthy/diseased, virulent/non-virulent) are run on the same gel they
can be directly compared. To do this with traditional 2D
electrophoresis requires large numbers of time-consuming repeats.
• In experiments comprising several gels, an internal standard in each
gel is included. The internal standard is prepared by mixing together
several or all of the samples in the experiment. This allows the
measurement of the abundance of a protein in each sample relative
to the internal standard. Since the amounts of each protein in the
internal standard are known to be the same in every gel, this method
reduces inter-gel variation
44. Isotachophoresis
• Used for separation of smaller ionic substances.
• They migrate adjacent in contact with one another, but not
overlapping.
• The sample is not mixed with the buffer prior to run.
• Hence current flow is carried entirely by the sample ions.
• Faster moving ions migrate first and the adjacent ones next with no
gap between the zone .
45. • All ions migrate at the rate of fastest ion in zones.
• Then it is measured by UV absorbance.
• Application
• Separation of small anions and cations
• Amino acids
• Peptides
• Nucleotides
• Nucleosides
• Proteins.
46. Capillary electrophoresis
Technique first described by- Jorgensen and Lukas (1980’s)
As the name suggest, the separation is carried in a narrow bore
Capillary tubes.
• 25-50μm – Internal diameter
• 300-330μm – External diameter
• Length – 50-100cm
• Fused silica capillary tube
• Polyimide coating external
• High voltage is applied (up to 50 kV)
48. Capillary electrophoresis
• The components migrate at different rate along the length
• Although separated by the electrophoretic migration, all the sample is
drawn towards cathode by electroendosmosis.
• Since this flow is strong, the rate of electroendosmotic flow is greater
than the Electrophoretic velocity of the analyte ion , regardless of the
charge.
• Positively charged molecule reach the cathode first (electrophoretic
migration + electrosmotic flow).
50. Sample application is done by either of one
method
High voltage injection
The buffer reservoir is
replaced by the sample
reservoir the high voltage
is applied buffer
reservoir is placed again
and voltage applied for the
separation.
Pressure injection
Capillary is removed from
buffer and placed in air tight
sample
with pressure sample is
pushed into capillary
capillary kept back in the
buffer sample and voltage is
applied for the separation.
51. Available menu include
• Serum protein
• Serum immunotyping
• Urine immunotyping
• Multiple myeloma testing
• Haemoglobinopathy screening
• HbA1c
53. Serum electrophoresis
Separates proteins into 5 different bands:
• Albumin, and Prealbumin. Albumin is formed in the liver and is 60%
of total proteins.
• alpha1-globulin (α1-globulin).
• alpha 2-globulin (α2-globulin).
• beta – globulin (β-globulin).
• gamma – globulin (γ-globulin).
54. Pattern of different bands on serum protein
electrophoresis
• Albumin zone shows only albumin.
• Alpha1- zone shows : alpha1 – lipoprotein , High-density lipoprotein
(HDL) , Alpha-1 -antitrypsin.
• Alpha 2 zone shows : Alpha-2 macroglobulin , Haptoglobin ,
β -lipoprotein.
• Beta zone shows : Transferrin , Complement 3 (C3).
• Gamma zone shows : Immunoglobulin(IgM, IgA, IgG) .
55. Troubleshooting
Adsorption of protein to the wall of capillary
– leading to smearing of protein
– viewed as peak broadening
– or complete loss of protein.
- Use of neutral coating group to the inner surface of the capillary.
56. Advantage over conventional
• Online detection.
• Improved quantification.
• Almost complete automation.
• Reduced analysis time.
57. Microchip electrophoresis
Current advanced method.
Development in technique include:
Integrated microchip design
Advanced detection system
New application
• Protein and DNA separation can be done
58. Instrumentation
Similar to the capillary electrophoresis.
• Separation channel
• Sampleinjection (50-100pL)
• Reservoirs
• Voltage (1-4kV)
• sample preparation
• Pre column or post column reactors.
• Classical Cross-T design.
• Time period of 50-200sec.
60. Thank you…
Ref..
• Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 8th
Edition
• Principles and Techniques of Biochemistry and Molecular Biology,
Wilson and Walker 8th Edition
• YouTube and Google images.
