Polyacrylamide gel electrophoresis

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Picture of an SDS-PAGE. The molecular markers (ladder) are in the left lane

Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule.

As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules' higher-order structure, or a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured linear chain whose mobility depends only on its length and mass-to-charge ratio. For nucleic acids, urea is the most commonly used denaturant. For proteins, sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein sample to linearize proteins and to impart a negative charge to linearized proteins. This procedure is called SDS-PAGE. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. Proteins that have a greater hydrophobic content, for instance many membrane proteins, and those that interact with surfactants in their native environment, are intrinsically harder to treat accurately using this method, due to the greater variability in the ratio of bound SDS.[1]

Procedure[edit]

Sample preparation[edit]

Samples may be any material containing proteins or nucleic acids. These may be biologically derived, for example from prokaryotic or eukaryotic cells, tissues, viruses, environmental samples, or purified proteins. In the case of solid tissues or cells, these are often first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), by sonicator or by using cycling of high pressure, and a combination of biochemical and mechanical techniques – including various types of filtration and centrifugation – may be used to separate different cell compartments and organelles prior to electrophoresis. Synthetic biomolecules such as oligonucleotides may also be used as analytes.

Reduction of a typical disulfide bond by DTT via two sequential thiol-disulfide exchange reactions.

The sample to analyze is optionally mixed with a chemical denaturant if so desired, usually SDS for proteins or urea for nucleic acids. SDS is an anionic detergent that denatures secondary and non–disulfide–linked tertiary structures, and additionally applies a negative charge to each protein in proportion to its mass. Urea breaks the hydrogen bonds between the base pairs of the nucleic acid, causing the constituent strands to separate. Heating the samples to at least 60 °C further promotes denaturation.[2][3][4][5]

In addition to 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). This is known as reducing SDS-PAGE.

A tracking dye may be added to the solution. This typically has a higher electrophoretic mobility than the analytes to allow the experimenter to track the progress of the solution through the gel during the electrophoretic run.

SDS-PAGE sample.png

Preparing acrylamide gels[edit]

The gels typically consist of acrylamide, bisacrylamide, the optional denaturant (SDS or urea), and a buffer with an adjusted pH. The solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization. Alternatively, butanol may be added to the resolving gel (for proteins) after it is poured, as butanol removes bubbles and makes the surface smooth. [6] A source of free radicals and a stabilizer, such as ammonium persulfate and TEMED are added to initiate polymerization.[7] The polymerization reaction creates a gel because of the added bisacrylamide, which can form cross-links between two acrylamide molecules. The ratio of bisacrylamide to acrylamide can be varied for special purposes, but is generally about 1 part in 35. 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 molecules, while much higher percentages are needed to resolve smaller proteins.

SDS-PAGE acrylamide stock.png

Gels are usually polymerized between two glass plates in a gel caster, with a comb inserted at the top to create the sample wells. After the gel is polymerized the comb can be removed and the gel is ready for electrophoresis.

SDS-PAGE Acrylamide gel.png

Electrophoresis[edit]

Various buffer systems are used in PAGE depending on the nature of the sample and the experimental objective. The buffers used at the anode and cathode may be the same or different.[8][9] [10]

SDS-PAGE Buffers.png
SDS-PAGE Electrophoresis.png

An electric field is applied across the gel, causing the negatively charged proteins or nucleic acids to migrate across the gel from the negative electrode (the cathode) towards the positive electrode (the anode). Depending on their size, each biomolecule moves differently through the gel matrix: small molecules more easily fit through the pores in the gel, while larger ones have more difficulty. The gel is run usually for a few hours, though this depends on the voltage applied across the gel; migration occurs more quickly at higher voltages, but these results are typically less accurate than at those at lower voltages. After the set amount of time, the biomolecules have migrated different distances based on their size. Smaller biomolecules travel farther down the gel, while larger ones remain closer to the point of origin. Biomolecules may therefore be separated roughly according to size, which depends mainly on molecular weight under denaturing conditions, but also depends on higher-order conformation under native conditions. However, certain glycoproteins behave anomalously on SDS gels.

Further processing[edit]

Two SDS-PAGE-gels after a completed run

Following electrophoresis, the gel may be stained (for proteins, most commonly with Coomassie Brilliant Blue R-250; for nucleic acids, ethidium bromide; or for either, silver stain), allowing visualization of the separated proteins, or processed further (e.g. Western blot). After staining, different species biomolecules appear as distinct bands within the gel. It is common to run molecular weight size markers of known molecular weight in a separate lane in the gel to calibrate the gel and determine the approximate molecular mass of unknown biomolecules by comparing the distance traveled relative to the marker.

For proteins, SDS-PAGE is usually the first choice as an assay of purity due to its reliability and ease. The presence of SDS and the denaturing step make proteins separate, approximately based on size, but aberrant migration of some proteins may occur. Different proteins may also stain differently, which interferes with quantification by staining. PAGE may also be used as a preparative technique for the purification of proteins. For example, quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) is a method for separating native metalloproteins in complex biological matrices.

Chemical ingredients and their roles[edit]

Polyacrylamide gel (PAG) had been known as a potential embedding medium for sectioning tissues as early as 1964, and two independent groups employed PAG in electrophoresis in 1959.[11][12] It possesses several electrophoretically desirable features that make it a versatile medium. It is a synthetic, thermo-stable, transparent, strong, chemically relatively inert gel, and can be prepared with a wide range of average pore sizes.[13] The pore size of a gel is determined by two factors, the total amount of acrylamide present (%T) (T = Total concentration of acrylamide and bisacrylamide monomer) and the amount of cross-linker (%C) (C = bisacrylamide concentration). Pore size decreases with increasing %T; with cross-linking, 5%C gives the smallest pore size. Any increase or decrease in %C from 5% increases the pore size, as pore size with respect to %C is a parabolic function with vertex as 5%C. This appears to be because of non-homogeneous bundling of polymer strands within the gel. This gel material can also withstand high voltage gradients, is amenable to various staining and destaining procedures, and can be digested to extract separated fractions or dried for autoradiography and permanent recording.

Components[edit]

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. This is known as Native PAGE. 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.

Chemicals for processing and visualization[edit]

PAGE of rotavirus proteins stained with Coomassie blue
Silver stained SDS Polyacrylamide gels

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

See also[edit]

References[edit]

  1. ^ Rath, Arianna and Glibowicka, Mira and Nadeau, Vincent G. and Chen, Gong and Deber, Charles M. (2009). "Detergent binding explains anomalous SDS-PAGE migration of membrane proteins". Proceedings of the National Academy of Sciences 106 (6): 1760–1765. doi:10.1073/pnas.0813167106. 
  2. ^ Shapiro AL, Viñuela E, Maizel JV Jr. (September 1967). "Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.". Biochem Biophys Res Commun. 28 (5): 815–820. doi:10.1016/0006-291X(67)90391-9. PMID 4861258. 
  3. ^ Weber K, Osborn M (August 1969). "The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis.". J Biol Chem. 244 (16): 4406–4412. PMID 5806584. 
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  5. ^ Caprette, David. "SDS-PAGE". Retrieved 27 September 2009. 
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  7. ^ "SDS-PAGE". Retrieved 12 September 2009. 
  8. ^ Laemmli UK (August 1970). "Cleavage of structural proteins during the assembly of the head of bacteriophage T4". Nature 227 (5259): 680–685. doi:10.1038/227680a0. PMID 5432063. 
  9. ^ Schägger H, von Jagow G (Nov 1987). "Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa". Anal Biochem 166 (2): 368–379. doi:10.1016/0003-2697(87)90587-2. PMID 2449095. 
  10. ^ Andrews. "SDS-PAGE". Retrieved 27 September 2009. 
  11. ^ Davis BJ, Ornstein L (1959). "A new high resolution electrophoresis method.". Delivered at the Society for the Study of Blood at the New York Academy of Medicine. 
  12. ^ Raymond S, Weintraub L. (1959). "Acrylamide gel as a supporting medium for zone electrophoresis.". Science 130 (3377): 711. doi:10.1126/science.130.3377.711. PMID 14436634. 
  13. ^ Rüchel R, Steere RL, Erbe EF (1978). "Transmission-electron microscopic observations of freeze-etched polyacrylamide gels". J Chromatogr. 166 (2): 563–575. doi:10.1016/S0021-9673(00)95641-3. 
  14. ^ Golgi C (1873). "Sulla struttura della sostanza grigia del cervello". Gazzetta Medica Italiana (Lombardia) 33: 244–246. 
  15. ^ Kerenyi L, Gallyas F (1973). "Über Probleme der quantitiven Auswertung der mit physikalischer Entwicklung versilberten Agarelektrophoretogramme". Clin. Chim. Acta 47 (3): 425–436. doi:10.1016/0009-8981(73)90276-3. PMID 4744834. 
  16. ^ Switzer RC 3rd, Merril CR, Shifrin S (Sep 1979). "A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels". Anal Biochem. 98 (1): 231–237. doi:10.1016/0003-2697(79)90732-2. PMID 94518. 
  17. ^ Hempelmann E, Schulze M, Götze O (1984). "Free SH-groups are important for the polychromatic staining of proteins with silver nitrat". Neuhof V (ed)Electrophoresis '84 , Verlag Chemie Weinheim 1984: 328–330. 
  18. ^ Grant G (Oct 2007). "How the 1906 Nobel Prize in Physiology or Medicine was shared between Golgi and Cajal". Brain Res Rev 55 (2): 490–498. doi:10.1016/j.brainresrev.2006.11.004. PMID 17306375. 
  19. ^ Minde DP (2012). "Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp". PLOS ONE 7 (10): e46147. doi:10.1371/journal.pone.0046147. PMC 3463568. PMID 23056252. 

External links[edit]