In the chemical process of electrophoresis, an electric charge in a solution flows in the direction of an opposing electrode. While studying blood proteins in the 1930s, Swedish biophysicist Arne Tisselius invented electrophoresis. The Novel Prize in Chemistry was awarded to Arne Tisselius in 1948 for his contributions to the electrophoretic method.
One of the laboratory techniques for separating DNA, RNA, or protein molecules according to their electric charge or size is gel electrophoresis.
Table of Contents
- Principle of Gel Electrophoresis
- Parts of Gel Electrophoresis Apparatus
- Buffers
- Container for staining and de-staining gel
- Polyacrylamide gel electrophoresis (PAGE)
- Pulsed-field gel electrophoresis (PFGE)
- Sodium dodecyl sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE)
- Immuno-electrophoresis (Rocket Electrophoresis)
- Difference Gel Electrophoresis (DIGE)
- Operating procedures of Electrophoresis
- Applications of Electrophoresis
- Advantages of Electrophoresis
- Limitations of Electrophoresis
- Examples of Electrophoresis System
- DNA electrophoresis system GEP-TH-1000TBT (Manufacturer: Bioevopeak)
- Electrofocusing electrophoresis system BT105 (Manufacturer: G BIOSCIENCES)
- DNA electrophoresis system EPS-2014 (Manufacturer: INOVIALAB)
- Isoelectric focusing electrophoresis system SymphonyIEF (Manufacturer: Hercuvan)
Principle of Gel Electrophoresis
The fact that the bulk of biomolecules exist as electrically charged particles with ionizable functional groups forms the basis of the electrophoresis principle. Depending on the pH, a solution containing biomolecules will have either positively or negatively charged ions.
Charged molecules move in the opposite direction of the positive or negative pole when they are exposed to an electric field. Ionized biomolecules will migrate at various speeds in the presence of an electric field, depending on the mass and net charge of each particle in the solution. Positively charged particles move toward the cathode, whereas negatively charged particles, such nucleic acids, migrate toward the anode. Changes in speed and direction cause each charged particle to move according to its unique attribute, allowing for the separation of biomolecule components with related properties.
Parts of Gel Electrophoresis Apparatus
Power supply
- Constant current, voltage, or power are required for electrophoresis.
- The migration speed should be kept up with a consistent power supply.
- The power source is connected to the gel box via lead cables in the colours red (anode/positively charged electrode) and black (cathode/negatively charged electrode).
- These cables transport the electric current from the power supply to the gel box.
- The dissolved ions thermally stir as a result of increased resistance caused by an increase in current.
- The equipment's water will evaporate more quickly.
- This will cause the ion concentration in the buffer to increase.
- The negatively charged DNA and RNA can go to the positively charged red wire at the front of the gel box by way of the black wire, which is linked to the back of the box.
Buffers
- The buffer establishes both the system's pH and the solute's electrical charge.
- The following characteristics define the perfect buffer:
- maintain the analyte's dissolving capacity
- throughout the study, maintain a steady buffering capacity.
- It shouldn't interfere with the ability to find the desired analytes.
- Obtain the proper distance between objects.
There are two types of buffers. Both an acidic and a basic buffer. Acidic buffers, such as citrate, acetate, formate, and phosphate, are used for a lower pH. To maintain high pH values, simple buffers like tric, borate, and tricine are used.
- The buffers have the same valency (ionic strength) and molality. They are therefore made up of monovalent ions.
- When not in use, the created buffers need to be properly cooled since they might operate as a conducive habitat for the development of germs.
- The cold buffer can be used in the method since it improves sample resolution and decreases solvent evaporation.
- In big volumes, the buffer can be reused up to four times; however, in smaller quantities, it can be discarded immediately away.
- The greater ionic strength of the buffer helps to provide a crisper resolution even if the considerable heat generated poses a significant danger of harming heat-labile compounds.
Support Media
- Starch, polyacrylamide, agarose, and the cellulose acetate membrane in the shape of sheets, slabs, and columns are examples of supporting media.
- It is a colloid with a water content of greater than 90%.
- In order to separate molecules, it works as a molecular sieve.
- It is permeable, allowing small molecules to flow through but not bigger ones.
- There must be electrical neutrality.
- Nowadays, agarose gel is mainly used as a support medium for electrophoresis.
1. Starch Gel
- It is the first electrophoresis gel media.
- It makes it easier to separate proteins according to their molecular size and charge-to-mass ratio.
- Boiling the starch granule solution in a buffer resulted in a colloidal suspension, which when allowed to cool sets as a semisolid gel as a result of the interweaving of the branched chains of amylopectin.
- The use of petroleum jelly prevents swelling and shrinkage.
- It is possible to produce sharp zones and great resolving power.
- It is not currently employed since gel preparation with repeatability is difficult.
2. Cellulose Acetate
- Cationic acetate electrophoresis was initially created when Kohn demonstrated how to separate the protein haemoglobin contained in red blood cells and to identify abnormal haemoglobin in blood serum.
- The cellulose in filter papers is acetylated to create cellulose acetate. Normal sites of acetylation are at C-3 and C-6 of the glucose ring. The holes of cellulose acetate are larger than those in polyacrylamide and agarose, two other popular electrophoretic matrices.
3. Agarose
- Agarose, a naturally occurring linear polymer made up of galactose and 3,6-anhydro-galactose chains, is present in agar recovered from red seaweeds.
- Agarose is stored as a dry powder, similar to agar.
- By combining the agarose powder with the proper solution buffer, heating it, and allowing it to cool to room temperature, agarose gel can be cast.
- The amount of agarose in the solution buffer regulates the gel's pore size.
- Agarose gels between 0.8% and 5% (W/V) are widely employed to differentiate between DNA and RNA molecules.
- Resolution is not as good as polyacrylamide gels.
- It creates stable gels, has a neutral charge, and has a low gelling temperature. As a result, it is regarded as the ideal substance for gel electrophoresis. It can be either liquid or solid.
4. Polyacrylamide
- The copolymerization of acrylamide monomers with the crosslinking agent N, N-methylene-bis-acrylamide (commonly known as "bis-acrylamide") results in a clear, translucent gel.
- Polyacrylamide gel pores are regulated by the concentration of acrylamide, which must be in proportion to its crosslinking agent.
- It usually takes a minimal quantity of acrylamide gel (3%–15%) to separate DNA and proteins.
- Proteins are separated under denatured circumstances according to their size using sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE), where a greater concentration of acrylamide gel (10%–20%) is generally utilised.
5. Electrophoresis chamber
- It is a plastic tank or container that is filled with a buffer to stop the flow of biomolecules.
- The migration process may easily be observed because to its transparent cover.
- It is connected to a power source.
6. Container for staining and de-staining gel
- Trays and containers can be used for gel staining and de-staining.
- Open-form boxes and closed boxes are also readily accessible.
- Typically, they are based on propylene.
- They are fitted snugly and are transparent.
- They are stain- and chemical-resistant.
Electrodes
- Due to their capacity to attract charges with opposing charges, the two platinum electrodes aid in the separation of molecules.
- An anode binds positive ions, whereas a cathode binds negative ions.
Gel Caster and Comb
- After being dissolved in the solvent, the gel is poured into a gel caster, which holds it and stores it inside the device.
- To position the wells for sample loading, a comb is used to arrange them.
Types of Electrophoresis
There are several types of gel electrophoresis, namely:
- Paper gel electrophoresis
- Agarose gel electrophoresis
- Polyacrylamide Gel Electrophoresis (PAGE)
- Pulse-field gel electrophoresis (PFGE)
- SDS- PAGE
- 2D- electrophoresis
- Immunoelectrophoresis (Rocket Electrophoresis)
- Difference Gel Electrophoresis (DIGE)
They may also be divided into two groups: native and denaturing. In native gel electrophoresis, RNA or proteins are maintained in their native form while moving across the gel. In contrast, denaturing gel electrophoresis reduces the RNA or protein to its linear form either before or during the electrophoresis process. By adding a reducing agent to the sample, gel, and/or buffer, this reduction is accomplished. This secondary structure is created as a result of the separation of the bonds inside the RNA or protein molecule.
1. Paper gel electrophoresis
- In clinical contexts, it is employed to evaluate serum and other physiological fluids.
- opacity and nontoxicity
- It is easy to store
- Adsorption of proteins
- poorly conducting
- Background staining
- The OH groups of cellulose bind to proteins and impede electrophoretic movements, which results in bands trailing and poor resolution.
2. Agarose gel electrophoresis
- The resolution of the electrophoresis depends on the agarose concentration.
- It works well for isolating DNA fragments with sizes between 100 and 20 kilobase pairs.
- It also pertains to protein separation via electrophoresis.
- Isoelectric focusing, which involves employing a low concentration of agarose gel, can be used to separate amphoteric molecules according to their isoelectric point.
3. Polyacrylamide gel electrophoresis (PAGE)
- Protein separation calls for a greater concentration than DNA separation, and vice versa. It is utilised at a concentration of up to 30% (pH range: 4-9.0).
- a higher level of accuracy and dependability in porosity.
- Its use in determining the molecular weight of DNA, sequencing DNA, examining DNA purity, analysing recombinant DNA molecules, separating RNA molecules, and determining the molecular weight of RNA are all examples.
4. Pulsed-field gel electrophoresis (PFGE)
- Shwartz and Cantor first presented this approach in 1984.
- By adjusting the direction and intensity of the electrical field between electrodes, DNA may be separated in an agarose gel.
- This method is used to isolate high molecular weight DNA with several megabases or even complete chromosomes.
- Because it generates exact findings that are easily reproducible, PFGE is used in numerous disciplines.
- It is used in research on the molecular biology of food-borne pathogens, monitoring the genetic stability of organisms used in fermentation, mapping applications like chromosome rearrangement detection, RFLP, and DNA fingerprinting, and identifying related strains in the event of hospital outbreaks, among other things.
5. Sodium dodecyl sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE)
- Initially known as the U.K. Laemmli Method after its British creator.
- Lower Separating Gel has smaller pores and a pH of 8, whereas Upper Stacking Gel has wider pores and a pH of 6.
- SDS-PAGE, which uses sodium dodecyl sulphate (SDS, also known as sodium lauryl sulphate) and polyacrylamide gel to separate proteins based on polypeptide chain length, essentially eliminates the impact of the structure and charge.
- SDS, a detergent present in the sample buffer, along with a few reducing agents cause harm to proteins' tertiary structure by rupturing their disulfide bonds.
- It is used to assess whether or not protein samples are pure and to compute the molecular weight of the protein.
Isoelectric point and Isoelectric focusing (IEF)
- When proteins have no net charge, the pH level known as the isoelectric point is reached (pI). The high-resolution method known as isoelectric focusing allows the separation of proteins by their isoelectric points within a continuous pH gradient (IEF). The outstanding resolving power allows separation of compounds with pI differences of about 0.01 pH units.
- It is employed to separate all amphoteric compounds, fractionate proteins, and identify isoenzymes.
6. 2D gel electrophoresis
- It was developed as a combination of the 2DGel, IEF, and SDS-PAGE processes and is used to evaluate complex protein mixtures.
- In the first phase, IEF separates the protein according to its charges, and in the second step, according to its mass.
- The IEF gel is placed horizontally inside the SDS-PAGE gel to conduct electrophoresis because SDS treatment renders the isolated protein on the gel negatively charged.
- The concentrated proteins on the pI are consequently split according to their molecular weights.
7. Immuno-electrophoresis (Rocket Electrophoresis)
- In the immune-electrophoresis (IEP) procedure, the protein antigen is first separated using electrophoresis in semi-solid medium, and precipitin is then produced by an immunodiffusion reaction with the antiserum.
- On a horizontal plate, suitable antibodies that are complementary to the test antigen to be assessed are dissolved in molten agar solution. Antigens are injected into the gel in the form of wells. At the alkaline pH, Ag gains a negative charge, travels in anode-downward motion, combines with Ab to form the Ag-Ab complex, and precipitates. Once the gel has been dyed with an appropriate dye like CBB, the immuno-precipitates will then show as arcs like rockets.
8. Difference Gel Electrophoresis (DIGE)
- It was developed to deal with the quantitative aspect of studies on differential expression and to overcome several problems with 2D-PAGE, such as analytical fluctuations.
- Up to three distinct protein samples can be labelled with fluorescent dyes that are size- and charge-matched in order to observe each protein sample independently ( for example, Cy3, Cy5, Cy2). Combining, loading, and putting the three samples through 2D electrophoresis.
Operating procedures of Electrophoresis
- Gel solution preparation: By dissolving it with hot water, a gel is created. The fluid is then put into a mould or caster after cooling to a more comfortable temperature.
- Gel casting: Once the gel has dried, wells may be made in it using a comb. The electrophoretic chamber is then filled with the gel. Buffer can only occupy up to one-third of the chamber's overall capacity.
- Sample preparation: Loading dye, such as ethidium bromide or a fluorescent tag, is introduced to the sample to give it colour and density.
- To see how the sample moves across the gel, the DNA is extracted, pre-processed, and added to a solution containing some common blue dye.
- Sample loading: The sample is inserted into the wells using a sterile micropipette.
- Electrophoresis: The negative and positive leads, which connect to the chamber and a power supply where the voltage is set, respectively. When the power supply is turned on, an electric field and negatively charged particles are produced. Because molecules tend toward electrodes with the opposite charge to their own, DNA that is negatively charged migrates toward the anode.
- Stopping electrophoresis, Staining, and Visualization: The dye is used to visually track the movement. The power source has been shut off. After the operation, the gel is dyed and seen with a gel imager. The logarithm of the molecular weight is used to determine the sizes of the sample fragments by comparing them to the standard.
Applications of Electrophoresis
- To investigate crime scenes and conduct paternity tests, DNA fingerprinting is used to isolate DNA fragments.
- finding genetic changes and proteins connected to health and disease
- For scientific applications, it is used in the detection and purification of nucleic acids and proteins.
- Finding infections in blood, other tissues, or sources like food is helpful.
- It makes it easier to identify and purify proteins or nucleic acids, which are commonly studied in more detail using DNA sequencing or mass spectrometry.
- It is employed in evolutionary research and blotting techniques for the analysis of macromolecules.
- It makes Polymerase Chain Reaction findings review easier (PCR).
- Electrophoresis is advantageous for both the production and development of vaccines.
- Taxonomy-DNA profiling is used to distinguish between species and evolutionary connections.
Advantages of Electrophoresis
- reasonably priced.
- establishes a relationship between outcomes that are comparable.
- Very simple to carry out
- can analyse DNA using any kind of proof.
- superior resolution
- a wide variety of pore diameters are available.
- Stable throughout a broad pH, temperature, and ionic strength range
- transparent to light
- chemically unactive
- both hydrophilicity and electrical neutrality
Limitations of Electrophoresis
1. Limited sample analysis
- Techniques such as in situ hybridization can be used to examine the expression of genes at each individual location of a tissue sample.
- Every brain region in a sample can be examined using ISH, whereas only a few regions can be examined using electrophoresis techniques.
2. Measurements are not precise
- Western blotting can effectively separate proteins with comparable molecular weights using gel electrophoresis.
- Using a technique known as 2D electrophoresis, it can also more accurately separate proteins.
- To determine the precise mass of proteins, mass spectroscopy must be performed after the protein has been purified.
3. A substantial starting sample required
- Proteins cannot be amplified, as DNA and RNA were able do before electrophoresis. As a result, the efficacy of this approach is diminished by the need for a large tissue sample, and instead, flow cytometry and immunohistochemistry are usually employed to examine the protein expression in specific cells.
4. Limited visualization facility
- Small hormones, neurotransmitters, and ions cannot be measured using electrophoresis.
- They don't completely respond to the electrophoresis preparation, often known as SDS-PAGE, for two reasons, and even if they did, they wouldn't be able to separate adequately since they are too small. They would hurriedly emerge from the gel's base.
5. Low throughput
- Low throughput in the sense that it takes a while to create data. The production of research data and the establishment of complex associations by electrophoresis is subpar compared to PCR and flow cytometry, which are massively parallel and serial procedures.
Precautions
- Use of nonconductive seats and flooring is encouraged (made of wood or plastics).
- When working near or adjacent to an electrophoresis system, stay away from unintentional grounding points and conductors (such sinks and other waste sources).
- Sample loading shouldn't be pushed too aggressively because doing so might damage wells.
- While making the gel, put on gloves, a face mask, and goggles.
- Consider taking the necessary measures before handling EtBr since it is mutagenic and carcinogenic.
Examples of Electrophoresis System
DNA electrophoresis system GEP-TH-1000TBT (Manufacturer: Bioevopeak)
Features:
- The system has a built-in high-current power supply that, by directly managing the current between the titanium anode and the stainless-steel cathode, can immediately achieve high efficiency and fast transfer.
- The fastest and most effective protein transfer from gel to membrane is made possible by the transfer system's seamless integration of traditional transfer technologies.
Electrofocusing electrophoresis system BT105 (Manufacturer: G BIOSCIENCES)
Features:
- solves gel leak problems without tape.
- For small and big wells, there are two combs available.
- supplied with a power source that may be changed.
- Easily operated and portable.
DNA electrophoresis system EPS-2014 (Manufacturer: INOVIALAB)
Features:
- For DNA and RNA electrophoresis, the EPS-2014 Mini Electrophoresis System has a small, innovative design.
- Current can only flow to the electrodes while the lid is open due to a magnetic sensor.
- While the system is running, if the lid is taken off or opened, the current is immediately turned off.
Isoelectric focusing electrophoresis system SymphonyIEF (Manufacturer: Hercuvan)
Features
- SymphonyIEF Isoelectric Focusing is a flexible device that can handle the majority of IEF requirements, from low-volume to high-throughput applications.
- It functions with horizontal precast IEF and PAGE gels when the electrode frame is fastened to the cooling plate directly.
