Citizendia
Your Ad Here

Picture of an SDS-PAGE. The molecular marker is in the left lane
Picture of an SDS-PAGE. The molecular marker is in the left lane

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used in biochemistry, genetics and molecular biology to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight as well as higher order protein folding, posttranslational modifications and other factors). Sodium lauryl sulfate ( SLS) or sodium dodecyl sulfate ( SDS or NaDS ( C 12 H 25 S[[oxygen O]]4 A Polyacrylamide Gel is a separation matrix used in electrophoresis of Biomolecules, such as Proteins or DNA fragments Electrophoresis is the most well-known electrokinetic phenomenon. Biochemistry is the study of the chemical processes in living Organisms It deals with the Structure and function of cellular components such as Genetics (from Ancient Greek grc-Latn genetikos, “genitive” and that from grc-Latn genesis, “origin” a discipline of Biology, is Molecular biology is the study of Biology at a molecular level Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl Protein folding is the physical process by which a Polypeptide folds into its characteristic and functional three-dimensional structure.

Contents

Procedure

The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. Sodium lauryl sulfate ( SLS) or sodium dodecyl sulfate ( SDS or NaDS ( C 12 H 25 S[[oxygen O]]4 Denaturation is a process in which Proteins or Nucleic acids lose their structure (tertiary structure by application of some external stress or compound for Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure. The isoelectric point (pI is the PH at which a particular Molecule or surface carries no net electrical charge. In Biochemistry, the primary structure of a biological molecule is the exact specification of its atomic composition and the chemical bonds connecting those atoms (including This is known as Native PAGE. Native Polyacrylamide gel Electrophoresis is an electrophoretic separation method typically used in Proteomics and metallomics. Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide.

SDS binds in a ratio of approximately 1. 4 g SDS per 1. 0 g protein (although binding ratios can vary from 1. 1-2. 2 g SDS/g protein), giving an approximately uniform mass:charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein. A tracking dye may be added to the protein solution to allow the experimenter to track the progress of the protein solution through the gel during the electrophoretic run.

Chemical ingredients and its roles

''Polyacrylamide gel (PAG)'' had been known as a potential embedding medium for sectioning tissues as early as 1954. Two independent groups: Davis and Raymond, employed PAG in electrophoresis in 1959. It possesses several electrophoretically desirable features that made it a versatile medium. Polyacrylamide gel separates protein molecules according to both size and charge. It is a synthetic gel, thermo-stable, transparent, strong, relatively chemically inert, can be prepared with a wide range of average pore sizes, can withstand high voltage gradients, feasible to various staining and destaining procedures and can be digested to extract separated fractions or dried for autoradiography and permanent recording. The term high voltage characterizes electrical circuits in which the voltage used is the cause of particular safety concerns and insulation requirements An autoradiograph is an image on an X-ray film or nuclear emulsion produced by the pattern of decay emissions (e DISC electrophoresis utilizes gels of different pore sizes. The name DISC was derived from the discontinuities in the electrophoretic matrix and coincidentally from the discoid shape of the separated zones of ions (Anbalagan, 1999). There are two layers of gel, namely stacking or spacer gel, and resolving or separating gel.

Stacking gel

The stacking gel is a large pore polyacrylamide gel (4%). This gel is prepared with Tris buffer pH 6. Tris is an abbreviation of the Organic compound known as tris(hydroxymethylaminomethane with the formula (HOCH23CNH2 pH is the measure of the acidity or alkalinity of a Solution. 8 of about 2 pH units lower than that of electrophoresis buffer. These conditions provide an environment for Kohlrausch reactions, as a result, proteins are concentrated to several fold and a thin starting zone of the order of 19 μm is achieved in a few minutes. Friedrich Wilhelm Georg Kohlrausch ( October 14, 1840 – January 17, 1910) was a German Physicist who investigated the This gel is cast over the resolving gel. The height of the stacking gel region is always maintained more than double the height and the volume of the sample to be applied.

Resolving gel

The resolving gel is a small pore polyacrylamide gel (3 - 30%). The Tris buffer used is of pH 8. 8. In this gel, macro molecules separate according to their size. In the present experiment, 8%, 10% and 12% Resolving gel were used for separating different range of proteins. 8% gel for 24 – 205 kD proteins, 10% gel for 14-205 kD proteins and 12% gel for 14-66 kD proteins

Chemical ingredients

Chemicals for processing and visualization

The following chemicals are used for processing of the gel and the protein samples visualized in it:

Reducing SDS-PAGE

Besides the addition of SDS, proteins may optionally be briefly heated to near boiling in the presence of a reducing agent, such as dithiothreitol (DTT) or 2-mercaptoethanol (beta-mercaptoethanol/BME), which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits). Dithiothreitol (DTT is the common name for a small-molecule Redox reagent known as Cleland's reagent. 2-Mercaptoethanol (also &beta-mercaptoethanol) is the Chemical compound with the formula HOCH2CH2SH This is known as reducing SDS-PAGE, and is most commonly used. Non-reducing SDS-PAGE (no boiling and no reducing agent) may be used when native structure is important in further analysis (e. g. enzyme activity, shown by the use of zymograms). Zymography is an electrophoretic technique based on SDS-PAGE, that includes a Substrate copolymerized with the Polyacrylamide gel for the detection of For example, quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) is a new method for separating native metalloproteins in complex biological matrices. QPNC-PAGE, or quantitative preparative native continuous polyacrylamide gel electrophoresis, is a high-resolution technique applied in Biochemistry and Bioinorganic In Biochemistry, a metalloprotein is a generic term for a Protein that contains a Metal cofactor.

Electrophoresis and staining

Two SDS-PAGE-gels after a completed run
Two SDS-PAGE-gels after a completed run

The denatured proteins are subsequently applied to one end of a layer of polyacrylamide gel submerged in a suitable buffer. A Polyacrylamide Gel is a separation matrix used in electrophoresis of Biomolecules, such as Proteins or DNA fragments An electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the anode. Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty (they encounter more resistance). Wiktionary (a Portmanteau of Wiki and Dictionary) is a multilingual, Web -based project to create a Free After a set amount of time (usually a few hours- though this depends on the voltage applied across the gel; higher voltages run faster but tend to produce somewhat poorer resolution), the proteins will have differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin. Thus proteins may be separated roughly according to size (and therefore, molecular weight). Following electrophoresis, the gel may be stained (most commonly with Coomassie Brilliant Blue or silver stain), allowing visualisation of the separated proteins, or processed further (e. Coomassie dyes (also known as Coomassie Brilliant Dyes are a family of dyes commonly used to stain proteins in Sodium dodecyl sulfate and blue native Polyacrylamide Silver staining is the use of Silver to stain Histologic sections This kind of staining is important especially to show Proteins (for example type III g. Western blot). The western blot (alternatively immunoblot) is an Analytical technique used to detect specific Proteins in a given sample of tissue homogenate or After staining, different proteins will appear as distinct bands within the gel. It is common to run "marker proteins" of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker. The gel is actually formed because the acrylamide solution contains a small amount, generally about 1 part in 35 of bisacrylamide, which can form cross-links between two polyacrylamide molecules. The ratio of acrylamide to bisacrylamide can be varied for special purposes. The acrylamide concentration of the gel can also be varied, generally in the range from 5% to 25%. Lower percentage gels are better for resolving very high molecular weight proteins, while much higher percentages are needed to resolve smaller proteins. Determining how much of the various solutions to mix together to make gels of particular acrylamide concentration can be done on line

Gel electrophoresis is usually the first choice as an assay of protein purity due to its reliability and ease. The presence of SDS and the denaturing step causes proteins to be separated solely based on size. False negatives and positives are possible. A co migrating contaminant can appear as the same band as the desired protein. This comigration could also cause a protein to run at a different position or to not be able to penetrate the gel. This is why it is important to stain the entire gel including the stacking section. Coomassie Brilliant Blue will also bind with less affinity to glycoproteins and fibrous proteins, which interferes with quantification (Deutscher 1990).

Buffer systems

Most protein separations are performed using a "discontinuous" buffer system that significantly enhances the sharpness of the bands within the gel. During electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus into a single sharp band. This occurs in a region of the gel that has larger pores so that the gel matrix does not retard the migration during the focusing or "stacking" event. Negative ions from the buffer in the tank then "outrun" the SDS-covered protein "stack" and eliminate the ion gradient so that the proteins subsequently separate by the sieving action in the lower, "resolving" region of the gel.

Many people continue to use a tris-glycine or "Laemmli" buffering system that stacks and resolves at a pH of ~8. pH is the measure of the acidity or alkalinity of a Solution. 3-9. 0. These pHs promote disulfide bond formation between cysteine residues in the proteins, especially when they are present at high concentrations because the pKa of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins. In Chemistry, a disulfide usually refers to the structural unit composed of a linked pair of sulfur atoms Not to be confused with Cystine, its oxidized dimer Cysteine (abbreviated as Cys or C) is an α- Amino acid with Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e. g. , bis-tris, pH 6. Bis-tris is an abbreviation of the Trivial name ( Bis(2-hydroxyethyl-imino-tris(hydroxymethyl-methane) for 2--2-(hydroxymethyl-13-propanediol. 5) and include reducing agents (e. g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers with lower pHs is that the acrylamide gel is more stable so the gels can be stored for long periods of time before use.

References

See also

External links

© 2009 citizendia.org; parts available under the terms of GNU Free Documentation License, from http://en.wikipedia.org
 Dapyx Software network: MP3 Explorer | Ebook Manager | Zenithic