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; 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 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. There is ongoing research in developing new ways of utilizing the knowledge associated with affinity electrophoresis to improve its functionality and speed, as well as attempts to improve established methods and tailor them towards performing specific tasks. A type of electrophoretic mobility shift assay, agarose gel electrophoresis is used to separate protein-bound amino acid complexes from free amino acids. Using a low voltage to minimize the risk for heat damage, electricity is run across an agarose gel; this technique utilizes a high voltage with a 0.5× Tris-borate buffer run across an agarose gel. This method differs from the traditional agarose gel electrophoresis by utilizing a higher voltage to facilitate a shorter run time as well as yield a higher band resolution. Other factors included in developing the technique of rapid agarose gel electrophoresis are gel thickness, the percentage of agarose within the gel.
Boronate affinity electrophoresis utilizes boronic acid infused acrylimide gels to purify NAD-RNA. This purification allows for researchers to measure the kinetic activity of NAD-RNA decapping enzymes. Affinity capillary electrophoresis utilizes a formulary approach in accordance with the theory of electromigration; this method utilizes the inter-molecular interactions found in a free solution. "Affinity probes" consisting of fluorophore-labeled molecules that will bind to target molecules are mixed with the sample being tested. This mixture and its subsequent complexes are separated through capillary electrophoresis; the principle behind this type of electrophoresis is the mobility of the target molecules being altered by inter-molecular interactions. Affinity-trap polyacrylamide gel electrophoresis has become one of the most popular methods of protein separation; this is not only due to its separation qualities, but because it can be used in conjunction with a variety of other analytic methods, such as mass spectrometry, western blotting.
This method utilizes a two-step approach. First, a protein sample is run through a polyacrylamide gel using electrophoresis; the sample is transferred to a different polyacrylamide gel where affinity probes are immobilized. The proteins that do not have affinity for the affinity probes pass through the affinity-trap gel, proteins with affinity for the probes will be "trapped" by the immobile affinity probes; these trapped proteins are visualized and identified using mass spectrometry after in-gel digestion. Phosphate affinity electrophoresis utilizes an affinity probe which consists of a molecule that binds to divalent phosphate ions in neutral aqueous solution, known as a "Phos-Tag"; this methods utilizes a separation gel made of an acrylamide-pendent Phos-Tag monomer, copolymerized. Phosphorylated proteins migrate in the gel compared to non-phosphorylated proteins; this technique gives the researcher the ability to observe the differences in the phosphorylation states of any given protein. Immunoelectrophoresis Comprehensive texts edited by Niels H. Axelsen in Scandinavian Journal of Immunology, 1975 Volume 4 Supplement
Deoxyribonucleic acid is a molecule composed of two chains that coil around each other to form a double helix carrying the genetic instructions used in the growth, development and reproduction of all known organisms and many viruses. DNA and ribonucleic acid are nucleic acids; the two DNA strands are known as polynucleotides as they are composed of simpler monomeric units called nucleotides. Each nucleotide is composed of one of four nitrogen-containing nucleobases, a sugar called deoxyribose, a phosphate group; the nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. The nitrogenous bases of the two separate polynucleotide strands are bound together, according to base pairing rules, with hydrogen bonds to make double-stranded DNA; the complementary nitrogenous bases are divided into two groups and purines. In DNA, the pyrimidines are cytosine. Both strands of double-stranded DNA store the same biological information.
This information is replicated as and when the two strands separate. A large part of DNA is non-coding, meaning that these sections do not serve as patterns for protein sequences; the two strands of DNA are thus antiparallel. Attached to each sugar is one of four types of nucleobases, it is the sequence of these four nucleobases along the backbone. RNA strands are created using DNA strands as a template in a process called transcription. Under the genetic code, these RNA strands specify the sequence of amino acids within proteins in a process called translation. Within eukaryotic cells, DNA is organized into long structures called chromosomes. Before typical cell division, these chromosomes are duplicated in the process of DNA replication, providing a complete set of chromosomes for each daughter cell. Eukaryotic organisms store most of their DNA inside the cell nucleus as nuclear DNA, some in the mitochondria as mitochondrial DNA, or in chloroplasts as chloroplast DNA. In contrast, prokaryotes store their DNA only in circular chromosomes.
Within eukaryotic chromosomes, chromatin proteins, such as histones and organize DNA. These compacting structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed. DNA was first isolated by Friedrich Miescher in 1869, its molecular structure was first identified by Francis Crick and James Watson at the Cavendish Laboratory within the University of Cambridge in 1953, whose model-building efforts were guided by X-ray diffraction data acquired by Raymond Gosling, a post-graduate student of Rosalind Franklin. DNA is used by researchers as a molecular tool to explore physical laws and theories, such as the ergodic theorem and the theory of elasticity; the unique material properties of DNA have made it an attractive molecule for material scientists and engineers interested in micro- and nano-fabrication. Among notable advances in this field are DNA origami and DNA-based hybrid materials. DNA is a long polymer made from repeating units called nucleotides.
The structure of DNA is dynamic along its length, being capable of coiling into tight loops and other shapes. In all species it is composed of two helical chains, bound to each other by hydrogen bonds. Both chains are coiled around the same axis, have the same pitch of 34 angstroms; the pair of chains has a radius of 10 angstroms. According to another study, when measured in a different solution, the DNA chain measured 22 to 26 angstroms wide, one nucleotide unit measured 3.3 Å long. Although each individual nucleotide is small, a DNA polymer can be large and contain hundreds of millions, such as in chromosome 1. Chromosome 1 is the largest human chromosome with 220 million base pairs, would be 85 mm long if straightened. DNA does not exist as a single strand, but instead as a pair of strands that are held together; these two long strands coil in the shape of a double helix. The nucleotide contains both a segment of the backbone of a nucleobase. A nucleobase linked to a sugar is called a nucleoside, a base linked to a sugar and to one or more phosphate groups is called a nucleotide.
A biopolymer comprising multiple linked nucleotides is called a polynucleotide. The backbone of the DNA strand is made from alternating sugar residues; the sugar in DNA is 2-deoxyribose, a pentose sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings; these are known as the 3′-end, 5′-end carbons, the prime symbol being used to distinguish these carbon atoms from those of the base to which the deoxyribose forms a glycosidic bond. When imagining DNA, each phosphoryl is considered to "belong" to the nucleotide whose 5′ carbon forms a bond therewith. Any DNA strand therefore has one end at which there is a phosphoryl attached to the 5′ carbon of a ribose and another end a
Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, transporting molecules from one location to another. Proteins differ from one another in their sequence of amino acids, dictated by the nucleotide sequence of their genes, which results in protein folding into a specific three-dimensional structure that determines its activity. A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides, containing less than 20–30 residues, are considered to be proteins and are called peptides, or sometimes oligopeptides; the individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, encoded in the genetic code.
In general, the genetic code specifies 20 standard amino acids. Shortly after or during synthesis, the residues in a protein are chemically modified by post-translational modification, which alters the physical and chemical properties, stability and the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can work together to achieve a particular function, they associate to form stable protein complexes. Once formed, proteins only exist for a certain period and are degraded and recycled by the cell's machinery through the process of protein turnover. A protein's lifespan covers a wide range, they can exist for years with an average lifespan of 1 -- 2 days in mammalian cells. Abnormal or misfolded proteins are degraded more either due to being targeted for destruction or due to being unstable. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in every process within cells.
Many proteins are enzymes that are vital to metabolism. Proteins have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signaling, immune responses, cell adhesion, the cell cycle. In animals, proteins are needed in the diet to provide the essential amino acids that cannot be synthesized. Digestion breaks the proteins down for use in the metabolism. Proteins may be purified from other cellular components using a variety of techniques such as ultracentrifugation, precipitation and chromatography. Methods used to study protein structure and function include immunohistochemistry, site-directed mutagenesis, X-ray crystallography, nuclear magnetic resonance and mass spectrometry. Most proteins consist of linear polymers built from series of up to 20 different L-α- amino acids. All proteinogenic amino acids possess common structural features, including an α-carbon to which an amino group, a carboxyl group, a variable side chain are bonded.
Only proline differs from this basic structure as it contains an unusual ring to the N-end amine group, which forces the CO–NH amide moiety into a fixed conformation. The side chains of the standard amino acids, detailed in the list of standard amino acids, have a great variety of chemical structures and properties; the amino acids in a polypeptide chain are linked by peptide bonds. Once linked in the protein chain, an individual amino acid is called a residue, the linked series of carbon and oxygen atoms are known as the main chain or protein backbone; the peptide bond has two resonance forms that contribute some double-bond character and inhibit rotation around its axis, so that the alpha carbons are coplanar. The other two dihedral angles in the peptide bond determine the local shape assumed by the protein backbone; the end with a free amino group is known as the N-terminus or amino terminus, whereas the end of the protein with a free carboxyl group is known as the C-terminus or carboxy terminus.
The words protein and peptide are a little ambiguous and can overlap in meaning. Protein is used to refer to the complete biological molecule in a stable conformation, whereas peptide is reserved for a short amino acid oligomers lacking a stable three-dimensional structure. However, the boundary between the two is not well defined and lies near 20–30 residues. Polypeptide can refer to any single linear chain of amino acids regardless of length, but implies an absence of a defined conformation. Proteins can interact with many types of molecules, including with other proteins, with lipids, with carboyhydrates, with DNA, it has been estimated. Smaller bacteria, such as Mycoplasma or spirochetes contain fewer molecules, on the order of 50,000 to 1 million. By contrast, eukaryotic cells are larger and thus contain much more pro
Water is a transparent, tasteless and nearly colorless chemical substance, the main constituent of Earth's streams and oceans, the fluids of most living organisms. It is vital for all known forms of life though it provides no calories or organic nutrients, its chemical formula is H2O, meaning that each of its molecules contains one oxygen and two hydrogen atoms, connected by covalent bonds. Water is the name of the liquid state of H2O at standard ambient pressure, it forms precipitation in the form of rain and aerosols in the form of fog. Clouds are formed from suspended droplets of its solid state; when finely divided, crystalline ice may precipitate in the form of snow. The gaseous state of water is water vapor. Water moves continually through the water cycle of evaporation, condensation and runoff reaching the sea. Water covers 71% of the Earth's surface in seas and oceans. Small portions of water occur as groundwater, in the glaciers and the ice caps of Antarctica and Greenland, in the air as vapor and precipitation.
Water plays an important role in the world economy. 70% of the freshwater used by humans goes to agriculture. Fishing in salt and fresh water bodies is a major source of food for many parts of the world. Much of long-distance trade of commodities and manufactured products is transported by boats through seas, rivers and canals. Large quantities of water and steam are used for cooling and heating, in industry and homes. Water is an excellent solvent for a wide variety of chemical substances. Water is central to many sports and other forms of entertainment, such as swimming, pleasure boating, boat racing, sport fishing, diving; the word water comes from Old English wæter, from Proto-Germanic *watar, from Proto-Indo-European *wod-or, suffixed form of root *wed-. Cognate, through the Indo-European root, with Greek ύδωρ, Russian вода́, Irish uisce, Albanian ujë; the identification of water as a substance Water is a polar inorganic compound, at room temperature a tasteless and odorless liquid, nearly colorless with a hint of blue.
This simplest hydrogen chalcogenide is by far the most studied chemical compound and is described as the "universal solvent" for its ability to dissolve many substances. This allows it to be the "solvent of life", it is the only common substance to exist as a solid and gas in normal terrestrial conditions. Water is a liquid at the pressures that are most adequate for life. At a standard pressure of 1 atm, water is a liquid between 0 and 100 °C. Increasing the pressure lowers the melting point, about −5 °C at 600 atm and −22 °C at 2100 atm; this effect is relevant, for example, to ice skating, to the buried lakes of Antarctica, to the movement of glaciers. Increasing the pressure has a more dramatic effect on the boiling point, about 374 °C at 220 atm; this effect is important in, among other things, deep-sea hydrothermal vents and geysers, pressure cooking, steam engine design. At the top of Mount Everest, where the atmospheric pressure is about 0.34 atm, water boils at 68 °C. At low pressures, water cannot exist in the liquid state and passes directly from solid to gas by sublimation—a phenomenon exploited in the freeze drying of food.
At high pressures, the liquid and gas states are no longer distinguishable, a state called supercritical steam. Water differs from most liquids in that it becomes less dense as it freezes; the maximum density of water in its liquid form is 1,000 kg/m3. The density of ice is 917 kg/m3. Thus, water expands 9% in volume as it freezes, which accounts for the fact that ice floats on liquid water; the details of the exact chemical nature of liquid water are not well understood. Pure water is described as tasteless and odorless, although humans have specific sensors that can feel the presence of water in their mouths, frogs are known to be able to smell it. However, water from ordinary sources has many dissolved substances, that may give it varying tastes and odors. Humans and other animals have developed senses that enable them to evaluate the potability of water by avoiding water, too salty or putrid; the apparent color of natural bodies of water is determined more by dissolved and suspended solids, or by reflection of the sky, than by water itself.
Light in the visible electromagnetic spectrum can traverse a couple meters of pure water without significant absorption, so that it looks transparent and colorless. Thus aquatic plants and other photosynthetic organisms can live in water up to hundreds of meters deep, because sunlight can reach them. Water vapour is invisible as a gas. Through a thickness of 10 meters or more, the intrinsic color of water is visibly turquoise, as its absorption spectrum has
Coulomb's law, or Coulomb's inverse-square law, is an experimental law of physics that quantifies the amount of force between two stationary, electrically charged particles. The electric force between charged bodies at rest is conventionally called electrostatic force or Coulomb force; the quantity of electrostatic force between stationary charges is always described by Coulomb’s law. The law was first published in 1785 by French physicist Charles-Augustin de Coulomb, was essential to the development of the theory of electromagnetism, maybe its starting point, because it was now possible to discuss quantity of electric charge in a meaningful way. In its scalar form, the law is: F = k e q 1 q 2 r 2, where ke is Coulomb's constant, q1 and q2 are the signed magnitudes of the charges, the scalar r is the distance between the charges; the force of the interaction between the charges is attractive if the charges have opposite signs and repulsive if like-signed. Being an inverse-square law, the law is analogous to Isaac Newton's inverse-square law of universal gravitation, but gravitational forces are always attractive, while electrostatic forces can be attractive or repulsive.
Coulomb's law can be used to derive Gauss's law, vice versa. The two laws are equivalent; the law has been tested extensively, observations have upheld the law on a scale from 10−16 m to 108 m. Ancient cultures around the Mediterranean knew that certain objects, such as rods of amber, could be rubbed with cat's fur to attract light objects like feathers. Thales of Miletus made a series of observations on static electricity around 600 BC, from which he believed that friction rendered amber magnetic, in contrast to minerals such as magnetite, which needed no rubbing. Thales was incorrect in believing the attraction was due to a magnetic effect, but science would prove a link between magnetism and electricity. Electricity would remain little more than an intellectual curiosity for millennia until 1600, when the English scientist William Gilbert made a careful study of electricity and magnetism, distinguishing the lodestone effect from static electricity produced by rubbing amber, he coined the New Latin word electricus to refer to the property of attracting small objects after being rubbed.
This association gave rise to the English words "electric" and "electricity", which made their first appearance in print in Thomas Browne's Pseudodoxia Epidemica of 1646. Early investigators of the 18th century who suspected that the electrical force diminished with distance as the force of gravity did included Daniel Bernoulli and Alessandro Volta, both of whom measured the force between plates of a capacitor, Franz Aepinus who supposed the inverse-square law in 1758. Based on experiments with electrically charged spheres, Joseph Priestley of England was among the first to propose that electrical force followed an inverse-square law, similar to Newton's law of universal gravitation. However, he did not elaborate on this. In 1767, he conjectured. In 1769, Scottish physicist John Robison announced that, according to his measurements, the force of repulsion between two spheres with charges of the same sign varied as x−2.06. In the early 1770s, the dependence of the force between charged bodies upon both distance and charge had been discovered, but not published, by Henry Cavendish of England.
In 1785, the French physicist Charles-Augustin de Coulomb published his first three reports of electricity and magnetism where he stated his law. This publication was essential to the development of the theory of electromagnetism, he used a torsion balance to study the repulsion and attraction forces of charged particles, determined that the magnitude of the electric force between two point charges is directly proportional to the product of the charges and inversely proportional to the square of the distance between them. The torsion balance consists of a bar suspended from its middle by a thin fiber; the fiber acts as a weak torsion spring. In Coulomb's experiment, the torsion balance was an insulating rod with a metal-coated ball attached to one end, suspended by a silk thread; the ball was charged with a known charge of static electricity, a second charged ball of the same polarity was brought near it. The two charged balls repelled one another, twisting the fiber through a certain angle, which could be read from a scale on the instrument.
By knowing how much force it took to twist the fiber through a given angle, Coulomb was able to calculate the force between the balls and derive his inverse-square proportionality law. Coulomb's law states that: The magnitude of the electrostatic force of attraction or repulsion between two point charges is directly proportional to the product of the magnitudes of charges and inversely proportional to the square of the distance between them; the force is along the straight line joining them. If the two charges have the same sign, the electrostatic force between them is repulsive. Coulomb's law can be stated as a simple mathematical expression; the scalar and vector forms of the mathematical equation are | F | = k e | q 1 q
Isotachophoresis 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 factor which tells how fast an ion migrates through an electric field. In conventional ITP separations, a discontinuous buffer system is used; the sample is introduced between a zone of fast leading electrolyte and a zone of slow terminating electrolyte. The LE and the TE have a common counterion, but the coions are different: the LE is defined by coions with high ionic mobility, while the TE is defined by coions with low ionic mobility; the analytes of interest have intermediate ionic mobility. Application of an electric potential results in a low electrical field in the leading electrolyte and a high electrical field in the terminating electrolyte. Analyte ions situated in the TE zone will migrate faster than the surrounding TE coions, while analyte ions situated in the LE will migrate slower.
ITP is a displacement method: focusing ions of a certain kind displace other ions. If present in sufficient amounts, focusing analyte ions can displace all electrolyte coions, reaching a plateau concentration. Multiple analytes with sufficiently different ionic mobilities will form multiple plateau zones. Indeed, plateau mode ITP separations are recognized by stairlike profiles, each plateau of the stair representing an electrolyte or analyte zone having increasing electric fields and decreasing conductivities. In peak mode ITP, analytes amounts are insufficient to reach plateau concentrations, such analytes will focus in sharp Gaussian-like peaks. In peak mode ITP, analyte peaks will overlap, unless so-called spacer compounds are added with intermediate ionic mobilities between those of the analytes. A completed ITP separation is characterized by a dynamic equilibrium in which all coionic zones migrate with equal velocities. From this phenomenon ITP has obtained its name: iso = equal, tachos = speed, phoresis = migration.
Isotachophoresis is equal to the steady-state-stacking step in Discontinuous electrophoresis. A popular form of ITP is transient ITP, it alleviates the limitation of conventional ITP that it has limited separation capacity because of analyte zone overlap. In transient ITP, analytes are first concentrated by ITP, can be baseline separated by zone electrophoresis. Transient ITP is accomplished by dissolving the sample in the TE and sandwiching the sample/TE plug between LE zones - or vice versa: a sample/LE plug can be sandwiched between TE zones. In the first case, analytes are focused at the front TE/LE interface. Meanwhile, the back of the TE plug becomes dissolved in the LE because the faster LE ions overcome the TE ions; when all of the TE ions are dissolved, the focusing process ceases and the analytes are separated according to the principles of zone electrophoresis. TITP is nowadays more widespread than conventional ITP because it is implemented in capillary electrophoresis separations as a preconcentration step, making CE more sensitive while profiting from its powerful separation capacities.
Adam, Albert. Biochemical and biological applications of isotachophoresis. Elsevier Scientific Pub. Co. ISBN 0-444-41891-1
Gel electrophoresis is a method for separation and analysis of macromolecules 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. 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 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 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, can act as a sieving medium, retarding the passage of molecules. DNA Gel electrophoresis is performed for analytical purposes after amplification of DNA via polymerase chain reaction, 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. In simple terms, electrophoresis is a process. Using an electric field, molecules can be made to move through a gel made of agarose or polyacrylamide; the electric field consists of a negative charge at one end which pushes the molecules through the gel, a positive charge at the other end that pulls the molecules through the gel. The molecules being sorted are dispensed into a well in the gel material; the gel is placed in an electrophoresis chamber, connected to a power source. When the electric current is applied, the larger molecules move more through the gel while the smaller molecules move faster.
The different sized molecules form distinct bands on the gel. The term "gel" in this instance refers to the matrix used to contain separate the target molecules. In most cases, the gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed; when separating proteins or small nucleic acids the gel is composed of different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids, the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled using appropriate safety precautions to avoid poisoning. Agarose is composed of long unbranched chains of uncharged carbohydrate without cross links resulting in a gel with large pores allowing for the separation of macromolecules and macromolecular complexes. Electrophoresis refers to the electromotive force, used to move the molecules through the gel matrix.
By placing the molecules in wells in the gel and applying an electric field, the molecules will move through the matrix at different rates, determined by their mass when the charge-to-mass ratio of all species is uniform. However, when charges are not all uniform the electrical field generated by the electrophoresis procedure will affect the species that have different charges and therefore will attract the species according to their charges being the opposite. Species that are positively charged will migrate towards the cathode, negatively charged. If the species are negatively charged they will migrate towards the positively charged anode. If several samples have been loaded into adjacent wells in the gel, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components.
Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which means they are the same size. There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size; the distance a band travels is inversely proportional to the logarithm of the size of the molecule. There are limits to electrophoretic techniques. Since passing current through a gel causes heating, gels may melt during electrophoresis. Electrophoresis is performed in buffer solutions to reduce pH changes due to the electric field, important because the charge of DNA and RNA depends on pH, but running for too long can exhaust the buffering capacity of the solution. There are limitations in determining the molecular weight by SDS-PAGE when trying to find the MW of an unknown protein.
There are certain biological variables that are difficult or impossible to minimize and can affect the elect