Discontinuous electrophoresis (colloquially disc electrophoresis [lower-alpha 1] ) is a type of polyacrylamide gel electrophoresis. It was developed by Ornstein and Davis. [2] [1] This method produces high resolution and good band definition. It is widely used technique for separating proteins according to size and charge. [3]
In this method, the gel is divided into two discontinuous parts, resolving and stacking gel, both have different concentrations of polyacrylamide. [4] The one with lower concentration is stacked on top of the one with higher concentration. Discontinuity is based on four parameters: gel structure, pH value of the buffer, ionic strength of the buffer, and the nature of the ions in the gel and electrode buffer. The electrode buffer contains glycine. Glycine has very low net charge at pH 6.8 of stacking gel, so it has low mobility. The proteins are separated according to the principle of isotachophoresis and form stacks in the order of mobility (stacking effect). Mobility depends on net charge, not on the size of the molecule. Proteins move towards anode slowly at constant speed till they reach limit of separation gel. Suddenly, frictional resistance increases but glycine is not affected and it passes the proteins and becomes highly charged in resolving zone. Proteins present in homogeneous buffer start to separate based on principles of zone electrophoresis. Now their mobility depends on size as well as charge. pH value rises to 9.5 and net charge increases. [5] [6]
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, 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.
Gel electrophoresis is a method for separation and analysis of biomacromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size 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.
The isoelectric point (pI, pH(I), IEP), is the pH at which a molecule carries no net electrical charge or is electrically neutral in the statistical mean. The standard nomenclature to represent the isoelectric point is pH(I). However, pI is also used. For brevity, this article uses pI. The net charge on the molecule is affected by pH of its surrounding environment and can become more positively or negatively charged due to the gain or loss, respectively, of protons (H+).
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.
In chemistry, electrophoresis is the motion of charged dispersed particles or dissolved charged molecules relative to a fluid under the influence of a spatially uniform electric field. As a rule, these are zwitterions. Electrophoresis of positively charged particles or molecules (cations) is sometimes called cataphoresis, while electrophoresis of negatively charged particles or molecules (anions) is sometimes called anaphoresis.
Polyacrylamide (abbreviated as PAM or pAAM) is a polymer with the formula (-CH2CHCONH2-). It has a linear-chain structure. PAM is highly water-absorbent, forming a soft gel when hydrated. In 2008, an estimated 750,000,000 kg were produced, mainly for water treatment and the paper and mineral industries.
Protein electrophoresis is a method for analysing the proteins in a fluid or an extract. The electrophoresis may be performed with a small volume of sample in a number of alternative ways with or without a supporting medium, namely agarose or polyacrylamide. Variants of gel electrophoresis include SDS-PAGE, free-flow electrophoresis, electrofocusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, counterelectrophoresis, and capillary electrophoresis. Each variant has many subtypes with individual advantages and limitations. Gel electrophoresis is often performed in combination with electroblotting or immunoblotting to give additional information about a specific protein.
Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis usually performed on proteins in a gel that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.
Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels. Very often, CE refers to capillary zone electrophoresis (CZE), but other electrophoretic techniques including capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis and micellar electrokinetic chromatography (MEKC) belong also to this class of methods. In CE methods, analytes migrate through electrolyte solutions under the influence of an electric field. Analytes can be separated according to ionic mobility and/or partitioning into an alternate phase via non-covalent interactions. Additionally, analytes may be concentrated or "focused" by means of gradients in conductivity and pH.
Isotachophoresis (ITP) is a technique in analytical chemistry used for selective separation and concentration of ionic analytes. It is a form of electrophoresis; charged analytes are separated based on ionic mobility, a quantity which tells how fast an ion migrates through an electric field.
MOPS (3-(N-morpholino)propanesulfonic acid) is a buffer introduced in the 1960s, one of the twenty Good's buffers. It is a structural analog to MES, and like MES, its structure contains a morpholine ring. HEPES is a similar pH buffering compound that contains a piperazine ring. With a pKa of 7.20, MOPS is an excellent buffer for many biological systems at near-neutral pH.
ACES is a chemical compound that is one of Good's buffers. It was developed in the 1960s to provide buffer solutions with pH ranging from 6.15-8.35 for use in various applications. With a pKa of 6.9, it is often used as a buffering agent in biological and biochemical research. It is a zwitterionic buffer with a useful buffering range of 6.1-7.5. The pioneering publication by Good and his co-workers described the synthesis and physical properties of ACES buffer.
Tricine is an organic compound that is used in buffer solutions. The name tricine comes from tris and glycine, from which it was derived. It is a white crystalline powder that is moderately soluble in water. It is a zwitterionic amino acid that has a pKa1 value of 2.3 at 25 °C, while its pKa2 at 20 °C is 8.15. Its useful buffering range of pH is 7.4-8.8. Along with bicine, it is one of Good's buffering agents. Good first prepared tricine to buffer chloroplast reactions.
A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore, when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments.
QPNC-PAGE, or QuantitativePreparativeNativeContinuousPolyacrylamideGel Electrophoresis, is a bioanalytical, one-dimensional, high-resolution and high-precision technique applied in biochemistry and bioinorganic chemistry to separate proteins quantitatively by isoelectric point and by continuous elution from a gel column.
Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. Cross electrophoresis, the first affinity electrophoresis method, was created by Nakamura et al. Enzyme-substrate complexes have been detected using cross electrophoresis. The methods include the so-called electrophoretic mobility shift assay, charge shift electrophoresis and affinity capillary electrophoresis. The methods are based on changes in the electrophoretic pattern of molecules through biospecific interaction or complex formation. The interaction or binding of a molecule, charged or uncharged, will normally change the electrophoretic properties of a molecule. Membrane proteins may be identified by a shift in mobility induced by a charged detergent. Nucleic acids or nucleic acid fragments may be characterized by their affinity to other molecules. The methods have been used for estimation of binding constants, as for instance in lectin affinity electrophoresis or characterization of molecules with specific features like glycan content or ligand binding. For enzymes and other ligand-binding proteins, one-dimensional electrophoresis similar to counter electrophoresis or to "rocket immunoelectrophoresis", affinity electrophoresis may be used as an alternative quantification of the protein. Some of the methods are similar to affinity chromatography by use of immobilized ligands.
The history of electrophoresis for molecular separation and chemical analysis began with the work of Arne Tiselius in 1931, while new separation processes and chemical analysis techniques based on electrophoresis continue to be developed in the 21st century. Tiselius, with support from the Rockefeller Foundation, developed the "Tiselius apparatus" for moving boundary electrophoresis, which was described in 1937 in the well-known paper "A New Apparatus for Electrophoretic Analysis of Colloidal Mixtures". The method spread slowly until the advent of effective zone electrophoresis methods in the 1940s and 1950s, which used filter paper or gels as supporting media. By the 1960s, increasingly sophisticated gel electrophoresis methods made it possible to separate biological molecules based on minute physical and chemical differences, helping to drive the rise of molecular biology. Gel electrophoresis and related techniques became the basis for a wide range of biochemical methods, such as protein fingerprinting, Southern blot, other blotting procedures, DNA sequencing, and many more.
Free-flow electrophoresis (FFE), also known as carrier-free electrophoresis, is a matrix-free, high-voltage electrophoretic separation technique. FFE is an analogous technique to capillary electrophoresis, with a comparable resolution, that can be used for scientific questions, where semi-preparative and preparative amounts of samples are needed. It is used to quantitatively separate samples according to differences in charge or isoelectric point by forming a pH gradient. Because of the versatility of the technique, a wide range of protocols for the separation of samples like rare metal ions, protein isoforms, multiprotein complexes, peptides, organelles, cells, DNA origami, blood serum and nanoparticles exist. The advantage of FFE is the fast and gentle separation of samples dissolved in a liquid solvent without any need of a matrix, like polyacrylamide in gel electrophoresis. This ensures a very high recovery rate since analytes do not adhere to any carrier or matrix structure. Because of its continuous nature and high volume throughput, this technique allows a fast separation of preparative amounts of samples with a very high resolution. Furthermore, the separations can be conducted under native or denaturing conditions.
SDS-PAGE is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. The combined use of sodium dodecyl sulfate and polyacrylamide gel eliminates the influence of structure and charge, and proteins are separated by differences in their size. At least up to 2012, the publication describing it was the most frequently cited paper by a single author, and the second most cited overall.
N,N′-Diallyl-L-tartardiamide (DATD) is a crosslinking agent for polyacrylamide gels, e.g., as used for SDS-PAGE. Compared to bisacrylamide gels, DATD gels have a stronger interaction with glass, and therefore are used in applications where the polyacrylamide gel acts as a "plug" structural component at the bottom of a gel electrophoresis apparatus, thereby preventing a weak discontinuous gel from sliding out from or otherwise moving within the apparatus. Unlike bisacrylamide-polyacrylamide gels, DATD-polyacrylamide gels can be conveniently dissolved using periodic acid due to the presence of viscinal diols in DATD. DATD is the slowest polyacrylamide crosslinker tested, and has can act as an inhibitor of polymerization at high concentrations.
