Gel electrophoresis: Introduction, Types, and Their applications
Types of gel electrophoresis
Gel electrophoresis contains supporting medium as gel and it is of various types as shown below-
- Paper gel electrophoresis
- Agarose gel electrophoresis
- PolyAcrylamide Gel Electrophoresis (PAGE)
- Pulsed-field gel electrophoresis (PFGE)
- SDS-PAGE
- 2D electrophoresis
- Immunoelectrophoresis(Rocket Electrophoresis)
- Difference Gel Electrophoresis(DIGE)
Paper gel electrophoresis
Used in clinical investigations of serum and other body fluids
Adsorbs proteins
Poor conductivity
Background staining
OH groups of cellulose bind with proteins and retard electrophoretic movements causing trailing of bands and poor resolution
Non-transparent, non-toxic
Can be stored easily
Agarose gel electrophoresis
The 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
1.5 200–3000
2.0 100–300
PolyAcrylamide Gel Electrophoresis (PAGE)
Used up to 3-30% concentration(pH range 4-9.0): lower concentration for DNA separation and higher concentration for protein separation
The high degree of reproducibility and precise porosity
Polymerization
Chemical /photopolymerization
The Effective Separation Range of Polyacrylamide Gels of Various Percent Acrylamide Monomer for Use With SDS-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
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
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 megabases, even whole chromosomes.
Different Types of Pulsed Field Gel Electrophoresis
Pulsed Field Gradient Gel Electrophoresis (PFGE)
Orthogonal Field Alternating Gel Electrophoresis (OFAGE)
Transverse Alternating Field Electrophoresis (TAFE)
Field Inversion Gel Electrophoresis (FIGE)
Contour Clamped homogeneous Electrophoresis (CHEF)
Crossed-field, Rotating Electric Field and Rotating Gel Electrophoresis (RGE)
PACE (programmable autonomously controlled electrodes)
Zero Integrated Field Electrophoresis
Simultaneous Tangential / Rectangular Inversion Decussate Electrophoresis
DR (dynamic regulation)
AFIGE (asymmetric field inversion gel electrophoresis)
Application
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
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
Applications
- To determine the molecular weight of proteins. (Plotting M.Wt Vs distance traveled)
- To fractionate protein subunits
- To assess the purity of protein samples (because of high resolving power).
Isoelectric Point
There is a pH at which there is no net charge on a protein; this is the isoelectric point (pI)
Isoelectric focusing(IEF)
High resolution can be achieved permitting separation of proteins differing only by 0.01 pI
Applications
- 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.
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
Immunoelectrophoresis(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.
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.
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.
Bibliography
- Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 5th Edition
- Principles and Techniques of Biochemistry and Molecular Biology, Wilson and Walker 7th Edition
- Short protocols in molecular biology, 4th edition
- Pulsed-field gel electrophoresis( A practical Guide )by Bruce Birren21.
- Molecular Biology. Editor: David Freifelder, 2nd ed, Publisher Jones and Bartlett Inc.