Electrophoresis is the movement of particles under spatially uniform electric field in a fluid. In the year 1807,Ferdinand Frederic Reuss observed clay particles dispersed in water migrating at a constant electric field.This was caused by a charged interface between the particle surface and the surrounding liquid. The rate of migration of particle was dependent on the strength of the field, on the net charge size and shape of the molecules and also on the ionic strength, viscosity and temperature of medium in which the molecules where moving.Electrophoresis is simple, fast and highly sensitive analytical technique.It is mostly used in analytical chemistry or medical biology to study the properties of a single charged species and as a separation technique.This technique provides the basis for the separation of molecules by charge or binding affinity,size for example, for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or protein molecules using an applied electric field in a gel matrix (dna gel box,protein gel),running gels or gel electrophoresis tray.One of the the main gel matrix used in electrophoresis is polyacrylamide and agarose gel.DNA gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA by PCR, but can be used as a preparative technique prior to the use of other methods such as mass spectrometry, PCR, RFLP, cloning, DNA sequencing(DNA testing) or Southern blotting, further characterization.
Principle of electrophoresis
The surface adsorbed sample strongly influences the suspended particles by applying an electrical surface charge to which an external electric field exerts an electrostatic Coulomb force.According to the double layer theory, all surface charges in fluids are shielded by a diffuse layer of ions that have the same absolute charge but an opposite sign with respect to the surface charge.The electric field also exerts a force on the ions in the diffuse layer whose direction is opposite to that acting on the surface charge. This force is not actually exerted on the particle, but on the ions in the diffuse layer at some distance from the particle surface, and some of it is transmitted by viscous stress to the particle surface.This part of the force is also called the electrophoretic deceleration force.If the electric field is applied and the charged particle to be analyzed moves uniformly through the diffuse layer, the resulting total force is zero:
Considering the resistivity of the moving particles due to the viscosity of the dispersant, the velocity of a dispersed particle in the case of low turbulence and moderate electrical charge E is simply proportional to the applied field, whereby the electrophoretic mobility μe remains defined as:
The best known and most widespread theory of electrophoresis was developed by Smoluchowski in 1903.
where εr is the dielectric constant of the dispersion, ε0 is the permittivity of free space (C² N–11 m–2), η is dynamic viscosity of the dispersion medium (Pa s), and ζ is zeta potential (i.e., the electrokinetic potential of the slipping plane in the double layer).
The Smoluchowski theory is very powerful because at any concentration it works for dispersed particles of any shape.Unfortunately, its validity is limited.For example, it follows from the fact that it does not include the Debye length k-1.Debye length must, however, be important for electrophoresis, as follows immediately from the figure on the right.Increasing double layer thickness (DL) leads to the removal of the point of delay from the particle surface.The thicker DL must be the smaller delay force.
Detailed theoretical analysis has shown that the Smoluchowski theory is only valid for sufficiently thin DL when the particle radius a is much higher than the Debye length:
This ” thin double layer ” model offers tremendous simplifications not just for the theory of electrophoresis but for many other electrokinetic theories.This model is valid for most aqueous systems because there are only a few nanometers of Debye length.It only breaks for nano-colloids with ionic strength near water.
Smoluchowski ‘s theory also neglects surface conductivity contributions. This is expressed as a small Dukhin number condition in modern theory.
In an effort to expand the scope of electrophoretic theories, the opposite asymptotic case was considered when the Debye length is greater than the particle radius:
In this ” thick double layer ” condition, Huckel predicted the following relationship for electrophoretic mobility:
This model can be useful for certain nanoparticles and non – polar fluids, where the Debye length is much greater than usual.There are several analytical theories that incorporate the conductivity of the surface and eliminate the restriction of a small number of Dukhin led by Overbeek and Booth.Modern, stringent theories valid for any zeta potential and often any aλ stem primarily from the theory of DukhinSemenikhin.These theories confirm the numerical solution of the problem provided by O’Brien and White in the thin double layer limit.
How to prepare and run standard agarose gel
Equipments and Supplies
The electrophoresis equipments (electrophoresis equipment list) and supplies required for agarose gel electrophoresis are relatively simple and includes the following
- Electrophoresis chamber and power supply(electrophoresis power supply)
- Gel casting trays (mostly agarose gel tank depending on the type of electrophoresis) available in various sizes and made of UV-transparent plastic. The open ends are closed with tape while the Gel is cast, then removed before electrophoresis.
- Sample combs where molten agarose is poured into sample gel wells.
- Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or TBE.
- Loading buffer containing something dense (e.g. glycerol) to allow the sample to ” fall ” into the sample wells, and one or two tracking dyes that migrate into the gel and allow visual monitoring or how far electrophoresis has taken place.
- Ethidium bromide, fluorescent dye used for nucleic acid staining. NOTE: Ethidium bromide is a known mutagen and should be treated as a hazardous chemical-therefore,wear gloves.
- Transilluminator (UV) used to visualize Ethidium brominated DNA in gels. Take note to always wear protective eyewear in transilluminator DNA to prevent UV light damage.
How to pour and run agarose gel electrophoresis
To pour a gel, agarose gel powder is mixed with the desired concentration of the electrophoresis buffer and heated in a microwave oven until it is completely melted.Ethidium bromide is most commonly added to gel (final concentration 0.5 μg / ml) to facilitate DNA visualization after electrophoresis.After cooling the solution to about 6 ° C, a casting tray containing a sample comb is poured and solidified at room temperature.
The comb is removed after the gel has solidified with care not to rip the bottom of the wells.The gel is inserted horizontally into the electrophoresis chamber and is just covered with buffer.Samples containing DNA mixed with loading buffers are then piped into sample wells, the lid and the power leads are placed on the device and a current is applied.You can confirm that the current flows by watching bubbles coming from the electrodes.DNA will migrate to the positive electrode, usually red colored.
The DNA distance migrated in the gel can be measured by visually monitoring the migration of tracking dyes. Bromophenol blue and xylene cyanol dyes migrate through agarose gel at roughly the same rate as two-stranded 300 and 4000 bp DNA fragments.
When adequate migration has taken place, DNA fragments are visualized with Ethidium bromide staining.This fluorescent dye intercalates between DNA and RNA bases.It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in an Ethidium bromide dilute solution.The gel is placed on an ultraviolet transilluminator to visualize DNA or RNA.
Linear DNA fragments migrate through agarose gel with mobility inversely proportional to their molecular weight log10.In other words, if you plot the distance from the well that DNA fragments migrated to the log10 of either their molecular weights or the number of base pairs, an roughly straight line appears.
Requirements for the pouring and running agarose gel electrophoresis
- Electrophoretic unit(electrophoresis set or electrophoresis kit)
- conical flask
- Measuring cylinder
- power pack
- TAE buffer micro tips (1X)
- Gel loading dye,
Procedure agarose gel electrophoresis
- For the preparation of 0.8 percent agarose gel, 0.14 g agarose should be dissolved in TAE 20 ml (1X).
- Boil the mixture until a clear solution is form or obtain
- Leave the solution at room temperature until the suspension reaches 40 – 45 ° C.
- Then add 2µl of 1% EtBr
- Seal the casting tray properly and place the combs at appropriate place
- Pour the gel and leave at room temperature for 45-50 minutes to solidify the gel.
- Fill the buffer tank with TAE (1X) so that the gel is dip
- Load 5 µl of the sample into the well by mixing with 1µl of 6X loading dye containing Bromophenol blue.
- Switch on the power supply at the rate of 5V/cm
- When the electrophoretic front reaches the bottom of the gel,switch off the power supply.
- Place the gel over transilluminator and observe under UV light
Types of Electrophoresis
- Affinity electrophoresis
The methods include electrophoresis mobility shift, electrophoresis load shift and capillary electrophoresis affinity.The methods are based on electrophoretic molecule pattern changes (mainly macromolecules) through biospecific interaction or complex formation.The interaction or binding of the loaded or unloaded molecule usually changes the electrophoretic properties of the molecule.Membrane proteins can be identified by a mobility shift induced by a loaded detergent.Nucleic acids or nucleic acid fragments can be distinguished from their affinity to other molecules.The methods used to estimate binding constants, such as lectin affinity electrophoresis or molecule characterization with specific features such as glycan content or ligand binding.For enzymes and other ligand-binding proteins, affinity electrophoresis similar to electrophoresis or ” rocket immunoelectrophoresis ” can be used as an alternative quantification of the protein.Some methods are similar to affinity chromatography using immobilized ligands.
2. Capillary electrophoresis
Capillary electrophoresis (CE) can be used to separate ionic species through their load and friction and hydrodynamic radius.In traditional electrophoresis, electrically charged analytes move under the influence of the electric field in a conductive liquid medium.The technique of capillary electrophoresis (CE) was introduced in the 1960s to separate species based on their size to load ratio within a small capillary filled with electrolyte.
Immunoelectrophoresis is a general name for a number of biochemistry separation and characterization methods for proteins based on electrophoresis and antibody reaction.All immunoelectrophoresis variants require immunoglobulins known as antibodies that react to the proteins to be separated or characterized.There are three types
- Rocket immunoelectrophoresis : is one-dimensional quantitative immunoelectrophoresis
- Fused rocket immunoelectrophoresis : It is a modification of one-dimensional quantitative immunoelectrophoresis used for detailed measurement of proteins in fractions from protein separation experiments.
- Affinity immunoelectrophoresis : is based on changes in the electrophoretic pattern of proteins through specific interaction or complex formation with other macromolecules or ligands
4. Pulsed field gel electrophoresis
Pulsed gel electrophoresis is a technique used to separate large deoxyribonucleic acid (DNA) molecules by applying an electric field to a gel matrix that changes direction periodically.
While small fragments can generally find their way through the gel matrix more easily than large DNA fragments, there is a threshold length above 30–50 kb where all large fragments run at the same rate and appear as a single large diffuse band in the gel.However, the various DNA lengths react at different rates with periodic changes in field direction.That is, larger DNA pieces will be slower to realign their load when the field direction changes and smaller pieces will be faster.Over the course of time, each band will start to separate more and more, even in very large lengths.This makes it possible to separate very large DNA parts using PFGE.
One of the most common methods of electrophoresis protein analysis is the use of Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.SDS is a detergent that denatures proteins by binding to hydrophobic regions and coats the linear protein sequence with a set of SDS molecules essentially.The SDS is negatively charged and therefore becomes the dominant burden of the complex.
The number of SDS molecules binding is simply proportional to the protein size.The weight ratio load should not change with size.In solution (water), all different sized proteins covered by SDS would be of approximately the same mobility in principle.Proteins are not running through water, Instead, they run through a polymer inert, polyacrylamide.The density and pore size of this polymer can vary by the way you make it (monomer concentration and cross – linking agent concentration).The size of the molecules that can pass through the matrix can therefore be varied.This determines which molecular weight range the gel has the highest resolution power.
6. Native Gels
Protein gels can also be run without SDS. These are called native gels, because one does not denature the protein purposely. The native load on the protein (divided by its mass) determines how quickly and in what direction the protein will travel.
7. Electrofocusing Gels
Another variation of gel electrophoresis is to pour a gel with a pH gradient from end to end.As the protein travels through this pH gradient, its different ionizable groups receive or lose protons.It will eventually find a pH where its load is zero and at that point it will be stuck (focused).
8. DNA Agarose Gels
A simple way to separate quite large DNA fragments by size is to use agarose gel.Agarose is another type of matrix used for many purposes (e.g. support for bacteria growth on plates).DNA doesn’t need a detergent because it has already been evenly spaced under negative phosphate groups.As with SDS-PAGE, the load ratio is constant.The separation results from the matrix itself, like SDS-PAGE.The sensitivity range can be varied by changing agarose density
DNA denaturing polyacrylamide gels (often referred to as gel sequencing)To view smaller DNA molecules with much higher resolution, people usually denature DNA through heat and run it through a thin polyacrylamide gel which is also kept close to the denaturing temperature.These gels usually contain additional teeth such as Urea.Two DNA pieces which differ in size from one base can be distinguished from each other in this way.
What are the applications of electrophoresis
- Gel electrophoresis is used in molecular biology, genetics, microbiology and biochemistry.
- The results can be quantitatively analyzed by visualizing the gel with UV light and gel image device.The image is recorded with a computer camera and the band or place of interest is measured and compared to standard or markers loaded on the same gel.
- Depending on the type of analysis being conducted, other techniques are often implemented in conjunction with gel electrophoresis results, which provide a wide range of field-specific applications.