Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell [1] and Klose [2] in 1975.
2-D electrophoresis begins with electrophoresis in the first dimension and then separates the molecules perpendicularly from the first to create an electropherogram in the second dimension. In electrophoresis in the first dimension, molecules are separated linearly according to their isoelectric point. In the second dimension, the molecules are then separated at 90 degrees from the first electropherogram according to molecular mass. Since it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis.[ citation needed ]
The two dimensions that proteins are separated into using this technique can be isoelectric point, protein complex mass in the native state, or protein mass.[ citation needed ]
The result of this is a gel with proteins spread out on its surface. These proteins can then be detected by a variety of means, but the most commonly used stains are silver and Coomassie brilliant blue staining. In the former case, a silver colloid is applied to the gel. The silver binds to cysteine groups within the protein. The silver is darkened by exposure to ultra-violet light. The amount of silver can be related to the darkness, and therefore the amount of protein at a given location on the gel. This measurement can only give approximate amounts, but is adequate for most purposes. Silver staining is 100x more sensitive than Coomassie brilliant blue with a 40-fold range of linearity. [3]
Molecules other than proteins can be separated by 2D electrophoresis. In supercoiling assays, coiled DNA is separated in the first dimension and denatured by a DNA intercalator (such as ethidium bromide or the less carcinogenic chloroquine) in the second. This is comparable to the combination of native PAGE/SDS-PAGE in protein separation.[ citation needed ]
 | This section may require cleanup to meet Wikipedia's quality standards. The specific problem is: Wait a second, isoelectric focusing also uses an IPG per its article, and it is also followed by SDS. What's the real difference, if any?(December 2023) |
A common technique is to use an Immobilized pH gradient (IPG) in the first dimension. This technique is referred to as IPG-DALT. The sample is first separated onto IPG gel (which is commercially available) then the gel is cut into slices for each sample which is then equilibrated in SDS-mercaptoethanol and applied to an SDS-PAGE gel for resolution in the second dimension. Typically IPG-DALT is not used for quantification of proteins due to the loss of low molecular weight components during the transfer to the SDS-PAGE gel. [4]
In quantitative proteomics, these tools primarily analyze bio-markers by quantifying individual proteins, and showing the separation between one or more protein "spots" on a scanned image of a 2-DE gel. Additionally, these tools match spots between gels of similar samples to show, for example, proteomic differences between early and advanced stages of an illness. Software packages include Delta2D (discontinued), ImageMaster (discontinued), Melanie, PDQuest (discontinued), SameSpots and REDFIN – among others.[ citation needed ] While this technology is widely utilized, the intelligence has not been perfected. For example, while PDQuest and SameSpots tend to agree on the quantification and analysis of well-defined well-separated protein spots, they deliver different results and analysis tendencies with less-defined less-separated spots. [5] Comparative studies have previously been published to guide researchers on the "best" software for their analysis. [6] Although typically used for standard gel electrophoresis, Sciugo can also be used for figure-creation and quantification.[ citation needed ]
Challenges for automatic software-based analysis include incompletely separated (overlapping) spots (less-defined or separated), weak spots / noise (e.g., "ghost spots"), running differences between gels (e.g., protein migrates to different positions on different gels), unmatched/undetected spots, leading to missing values, [7] [8] mismatched spots, errors in quantification (several distinct spots may be erroneously detected as a single spot by the software and parts of a spot may be excluded from quantification), and differences in software algorithms and therefore analysis tendencies
Generated picking lists can be exported from some software packages [9] and used for the automated in-gel digestion of protein spots, and subsequent identification of the proteins by mass spectrometry. Mass spectrometry analysis can identify precise mass measurements along with the sequencing of peptides that range from 1000–4000 atomic mass units. [10] For an overview of the current approach for software analysis of 2DE gel images, see Berth et al. [11] or Bandow et al. [12]
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+).
Proteomics is the large-scale study of proteins. Proteins are vital macromolecules of all living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In addition, other kinds of proteins include antibodies that protect an organism from infection, and hormones that send important signals throughout the body.
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.
The western blot, or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination.
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.
Coomassie brilliant blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie brilliant blue G-250 differs from Coomassie brilliant blue R-250 by the addition of two methyl groups. The name "Coomassie" is a registered trademark of Imperial Chemical Industries.
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.
Peptide mass fingerprinting (PMF), also known as protein fingerprinting, is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer such as MALDI-TOF or ESI-TOF. The method was developed in 1993 by several groups independently. The peptide masses are compared to either a database containing known protein sequences or even the genome. This is achieved by using computer programs that translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides, and calculate the absolute masses of the peptides from each protein. They then compare the masses of the peptides of the unknown protein to the theoretical peptide masses of each protein encoded in the genome. The results are statistically analyzed to find the best match.
Difference gel electrophoresis (DIGE) is a form of gel electrophoresis where up to three different protein samples can be labeled with size-matched, charge-matched spectrally resolvable fluorescent dyes prior to two dimensional gel electrophoresis.
Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies. All variants of immunoelectrophoresis require immunoglobulins, also known as antibodies, reacting with the proteins to be separated or characterized. The methods were developed and used extensively during the second half of the 20th century. In somewhat chronological order: Immunoelectrophoretic analysis, crossed immunoelectrophoresis, rocket-immunoelectrophoresis, fused rocket immunoelectrophoresis ad modum Svendsen and Harboe, affinity immunoelectrophoresis ad modum Bøg-Hansen.
QPNC-PAGE, or QuantitativePreparativeNativeContinuousPolyacrylamideGel Electrophoresis, is a bioanalytical, one-dimensional, high-resolution and high-precision electrophoresis technique applied in biochemistry and bioinorganic chemistry to separate proteins quantitatively by isoelectric point and by continuous elution from a gel column.
Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. It can also be used to localize proteins to the various organelles, and determine the interactions between different proteins as well as with membrane lipids.
Top-down proteomics is a method of protein identification that either uses an ion trapping mass spectrometer to store an isolated protein ion for mass measurement and tandem mass spectrometry (MS/MS) analysis or other protein purification methods such as two-dimensional gel electrophoresis in conjunction with MS/MS. Top-down proteomics is capable of identifying and quantitating unique proteoforms through the analysis of intact proteins. The name is derived from the similar approach to DNA sequencing. During mass spectrometry intact proteins are typically ionized by electrospray ionization and trapped in a Fourier transform ion cyclotron resonance, quadrupole ion trap or Orbitrap mass spectrometer. Fragmentation for tandem mass spectrometry is accomplished by electron-capture dissociation or electron-transfer dissociation. Effective fractionation is critical for sample handling before mass-spectrometry-based proteomics. Proteome analysis routinely involves digesting intact proteins followed by inferred protein identification using mass spectrometry (MS). Top-down MS (non-gel) proteomics interrogates protein structure through measurement of an intact mass followed by direct ion dissociation in the gas phase.
Within chemistry for acid–base reactions, Immobilized pH gradient (IPG) gels are the acrylamide gel matrix co-polymerized with the pH gradient, which result in completely stable gradients except the most alkaline (>12) pH values. The immobilized pH gradient is obtained by the continuous change in the ratio of Immobilines. An Immobiline is a weak acid or base defined by its pK value. Immobilized pH gradients (IPG) are made by mixing two kinds of acrylamide mixture, one with Immobiline having acidic buffering property and other with basic buffering property. The concentrations of the buffers in the two solutions define the range and shape of the pH gradient produced. Both solutions contain acrylamide monomers and catalysts. During polymerization, the acrylamide portion of the buffers co polymerize with the acrylamide and bisacrylamide monomers to form a polyacrylamide gel. These polymerised gels are backed with plastic based backing that allow ease in handling and improve IPG's performance. The gel is then washed to remove catalysts and unpolymerized monomers, which interfere with isoelectric separation. IPG increased reproducibility of isoelectric focusing and 2D-gel electrophoresis. Other advantages are increased resolution, reproducible separation of alkaline proteins and increased loading capacity.
An electrophoretic color marker is a chemical used to monitor the progress of agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) since DNA, RNA, and most proteins are colourless. The color markers are made up of a mixture of dyes that migrate through the gel matrix alongside the sample of interest. They are typically designed to have different mobilities from the sample components and to generate colored bands that can be used to assess the migration and separation of sample components.
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.
A gel doc, also known as a gel documentation system, gel image system or gel imager, refers to equipment widely used in molecular biology laboratories for the imaging and documentation of nucleic acid and protein suspended within polyacrylamide or agarose gels. Genetic information is stored in DNA. Polyacrylamide or agarose gel electrophoresis procedures are carried out to examine nucleic acids or proteins in order to analyze the genetic data. For protein analysis, two-dimensional gel electrophoresis is employed (2-DGE) which is one of the methods most frequently used in comparative proteomic investigations that can distinguish thousands of proteins in a single run. Proteins are separated using 2-DGE first, based on their isoelectric points (pIs) in one dimension and then based on their molecular mass in the other. After that, a thorough qualitative and quantitative analysis of the proteomes is performed using gel documentation with software image assessment methods on the 2-DGE gels stained for protein visibility. Gels are typically stained with Ethidium bromide or other nucleic acid stains such as GelGreen.
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.
