Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF agarose, essentially size independent) and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.
- Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a matrix of agarose or other substances.
- Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.
- Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins. Gel electrophoresis can also be used for separation of nanoparticles.
- Gel electrophoresis uses a gel as an anticonvective medium or sieving medium during electrophoresis, the movement of a charged particle in an electrical field.
- Gels suppress the thermal convection caused by application of the electric field, and can also act as a sieving medium, retarding the passage of molecules; gels can also simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied.
- DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via polymerase chain reaction (PCR), but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.
Agarose gel electrophoresis
- Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar.
- The proteins may be separated by charge and/or size (isoelectric focusing agarose electrophoresis is essentially size independent), and the DNA and RNA fragments by length.
- Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.
Properties of agarose gel
- Agarose gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled bundles that are aggregated into three-dimensional structures with channels and pores through which biomolecules can pass.
- The 3-D structure is held together with hydrogen bonds and can therefore be disrupted by heating back to a liquid state.
- The melting temperature is different from the gelling temperature, depending on the sources, agarose gel has a gelling temperature of 35–42 °C and a melting temperature of 85–95 °C. Low-melting and low-gelling agaroses made through chemical modifications are also available.
- Agarose gel has large pore size and good gel strength, making it suitable as an anticonvection medium for the electrophoresis of DNA and large protein molecules.
- The pore size of a 1% gel has been estimated from 100 nm to 200–500 nm, and its gel strength allows gels as dilute as 0.15% to form a slab for gel electrophoresis. Low-concentration gels (0.1–0.2%) however are fragile and therefore hard to handle.
- Agarose gel has lower resolving power than polyacrylamide gel for DNA but has a greater range of separation, and is therefore used for DNA fragments of usually 50–20,000 bp in size.
- The agarose polymer contains charged groups, in particular pyruvate and sulphate.
- These negatively charged groups create a flow of water in the opposite direction to the movement of DNA in a process called electroendosmosis (EEO), and can therefore retard the movement of DNA and cause blurring of bands.
Buffers
- In general, the ideal buffer should have good conductivity, produce less heat and have a long life.
- There are a number of buffers used for agarose electrophoresis; common ones for nucleic acids include Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE).
- The buffers used contain EDTA to inactivate many nucleases which require divalent cation for their function.
- The borate in TBE buffer can be problematic as borate can polymerize, and/or interact with cis diols such as those found in RNA.
- TAE has the lowest buffering capacity, but it provides the best resolution for larger DNA.
- This means a lower voltage and more time, but a better product.
- Electrophoresis is performed in buffer solutions to reduce pH changes due to the electric field, which is important because the charge of DNA and RNA depends on pH, but running for too long can exhaust the buffering capacity of the solution.
- Further, different preparations of genetic material may not migrate consistently with each other, for morphological or other reasons.
General procedure
- 1. Casting of gel: The gel is prepared by dissolving the agarose powder in an appropriate buffer, such as TAE or TBE, to be used in electrophoresis. The agarose is dispersed in the buffer before heating it to near-boiling point, but avoid boiling. The melted agarose is allowed to cool sufficiently before pouring the solution into a cast as the cast may warp or crack if the agarose solution is too hot. A comb is placed in the cast to create wells for loading sample, and the gel should be completely set before use.
- 2. Loading of samples: Once the gel has set, the comb is removed, leaving wells where DNA samples can be loaded. Loading buffer is mixed with the DNA sample before the mixture is loaded into the wells.
- 3. Electrophoresis: Agarose gel electrophoresis is most commonly done horizontally in a submarine mode whereby the slab gel is completely submerged in buffer during electrophoresis. It is also possible, but less common, to perform the electrophoresis vertically, as well as horizontally with the gel raised on agarose legs using an appropriate apparatus. The buffer used in the gel is the same as the running buffer in the electrophoresis tank, which is why electrophoresis in the submarine mode is possible with agarose gel.
- 4. Staining and visualization: DNA as well as RNA are normally visualized by staining with ethidium bromide, which intercalates into the major grooves of the DNA and fluoresces under UV light. The intercalation depends on the concentration of DNA and thus, a band with high intensity will indicate a higher amount of DNA compared to a band of less intensity. The ethidium bromide may be added to the agarose solution before it gels, or the DNA gel may be stained later after electrophoresis. Destaining of the gel is not necessary but may produce better images. Other methods of staining are available; examples are SYBR Green, GelRed, methylene blue, brilliant cresyl blue, Nile blue sulphate, and crystal violet.
Applications
- Estimation of the size of DNA molecules following digestion with restriction enzymes, e.g. in restriction mapping of cloned DNA.
- Analysis of products of a polymerase chain reaction (PCR), e.g. in molecular genetic diagnosis or genetic fingerprinting
- Separation of DNA fragments for extraction and purification.
- Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.
- Separation of proteins, for example, screening of protein abnormalities in clinical chemistry.
Advantages
- Agarose gels are easily cast and handled compared to other matrices and nucleic acids are not chemically altered during electrophoresis.
- Samples are also easily recovered. After the experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator.
Disadvantages
- The disadvantages are that gels can melt during electrophoresis.
- The buffer can become exhausted, and different forms of genetic material may run in unpredictable forms.
Polyacrylamide gel electrophoresis
- Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility.
- Electrophoretic mobility is a function of the length, conformation and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples.
- When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.
Properties of Polyacrylamide gel
- Hydration of acrylonitrile results in formation of acrylamide molecules(C3H5NO)by nitrilehydratase.
- Acrylamide monomer is in a powder state before addition of water.
- Acrylamide is toxic to the human nervous system, therefore all safety measures must be followed when working with it.
- Acrylamide is soluble in water and upon addition of water it polymerizes resulting in formation of polyacrylamide.
- It is useful to make polyacrylamide gel via acrylmide hydration because pore size can be regulated.
- Increased concentrations of acrylamide result in decreased pore size after polymerization.
- Polyacrylamide gel with small pores helps to examine smaller molecules better since the small molecules can enter the pores and travel through the gel while large molecules get trapped at the pore openings.
- Polyacrylamide gels are composed of a stacking gel and separating gel.
- Stacking gels have a higher porosity relative to the separating gel, and allow for proteins to migrate in a concentrated area.
- Additionally, stacking gels usually have a pH of 6.8, since the neutral glycine molecules allow for faster protein mobility.
- Separating gels have a pH of 8.8, where the anionic glycine slows down the mobility of proteins.
- Separating gels allow for the separation of proteins and have a relatively lower porosity. Here, the proteins are separated based on size (in SDS-PAGE) and size/ charge (Native PAGE)
Types of PAGE
1)Native-PAGE.
- As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules’ higher-order structure. This method is called native-PAGE.
2)SDS-PAGE
- Alternatively, a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured molecule whose mobility depends only on its length (because the protein-SDS complexes all have a similar mass-to-charge ratio).
- This procedure is called SDS-PAGE. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a method of separating molecules based on the difference of their molecular weight.
- At the pH at which gel electrophoresis is carried out the SDS molecules are negatively charged and bind to proteins in a set ratio, approximately one molecule of SDS for every 2 amino acids.
- In this way, the detergent provides all proteins with a uniform charge-to-mass ratio. By binding to the proteins the detergent destroys their secondary, tertiary and/or quaternary structure denaturing them and turning them into negatively charged linear polypeptide chains.
Applications of Polyacrylamide Gel Electrophoresis (PAGE)
- Measuring molecular weight.
- Peptide mapping.
- Estimation of protein size.
- Determination of protein subunits or aggregation structures.
- Estimation of protein purity.
- Protein quantitation.
- Monitoring protein integrity.
- Comparison of the polypeptide composition of different samples.
- Analysis of the number and size of polypeptide subunits.
- Post-electrophoresis applications, such as Western blotting.
- Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid.
- Pouring and Running a Protein Gel by reusing Commercial Cassettes.
- Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes.
- Detection of Protein Ubiquitination.
Advantages of PAGE
- Stable chemically cross-linked gel
- Greater resolving power (Sharp bands)
- Can accommodate larger quantities of DNA without significant loss in resolution
- The DNA recovered from polyacrylamide gels is extremely pure
- The pore size of the polyacrylamide gels can be altered in an easy and controllable fashion by changing the concentrations of the two monomers.
- Good for separation of low molecular weight fragments
Disadvantages of PAGE
- Generally more difficult to prepare and handle, involving a longer time for preparation than agarose gels.
- Toxic monomers
- Gels are tedious to prepare and often leak
- Need new gel for each experiment Stable chemically cross-linked gel
