The pioneering work on protein electrophoresis by Arne Tiselius (for which he received the Nobel Prize in Chemistry in 1948) was performed in free solution. However, it was soon realised that many of the problems associated with this approach, particularly the adverse effects of diffusion and convection currents, could be minimised by stabilising the medium. This was achieved by carrying out electrophoresis on a porous mechanical support, which was wetted in electrophoresis buffer and in which electrophoresis of buffer ions and samples could occur. The support medium cuts down convection currents and diffusion so that the separated components remain as sharp zones. The earliest supports used were filter paper or cellulose acetate strips, wetted in electrophoresis buffer. Nowadays, these media are infrequently used, although cellulose acetate still has its uses. In particular, for many years small molecules such as amino acids, peptides and carbohydrates were routinely separated and analysed by electrophoresis on supports such as paper or thin-layer plates of cellulose, silica or alumina. Although occasionally still used nowadays, such molecules are now more likely to be analysed by more modern and sensitive techniques such as high-performance liquid chromatography ( HPLC). While paper or thin-layer supports are fi ne for resolving small molecules, the separation of macro molecules such as proteins and nucleic acids on such supports is poor.

However, the introduction of the use of gels as a support medium led to a rapid improvement in methods for analysing macromolecules. The earliest gel system to be used was the starch gel and, although this still has some uses, the vast majority of electrophoretic techniques used nowadays involve either agarose gels or polyacrylamide gels.

Agarose Gels

Agarose is a linear polysaccharide (average relative molecular mass about 12 000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose ( Figure 1). Agarose is one of the components of agar, which is a mixture of polysaccharides isolated from certain seaweeds. Agarose is usually used at concentrations of between 1% and 3%. Agarose gels are formed by suspending dry agarose in aqueous buffer, then boiling the mixture until a clear solution forms. This is poured and allowed to cool to room temperature to form a rigid gel. The gelling properties are attributed to both inter- and intramolecular hydrogen bonding within and between the long agarose chains. This cross-linked structure gives the gel good anticonvectional properties. The pore size in the gel is controlled by the initial concentration of agarose; large pore sizes are formed from low concentrations and smaller pore sizes are formed from the higher concentrations. Although essentially free from charge, substitution of the alternating sugar residues with carboxyl, methyoxyl, pyruvate and especially sulfate groups occurs to varying degrees. This substitution can result in electroendosomosis during electrophoresis and ionic interactions between the gel and sample in all uses, both unwanted effects. Agarose is therefore sold in different purity grades, based on the sulfate concentration – the lower the sulfate content, the higher the purity.

Fig1. Agarobiose, the repeating unit of agarose.

Agarose gels are used for the electrophoresis of both proteins and nucleic acids. For proteins, the pore sizes of a 1% agarose gel are large relative to the sizes of proteins. Agarose gels are therefore used in techniques such as flat-bed isoelectric focussing, where the proteins are required to move unhindered in the gel matrix according to their native charge. Such large pore gels are also used to separate much larger molecules such as DNA or RNA, because the pore sizes in the gel are still large enough for DNA or RNA molecules to pass through the gel. In these cases, however, the pore size and molecule size are more comparable and fractional effects begin to play a role in the separation of these molecules. A further advantage of using agarose is the availability of low melting temperature agarose (62–65 °C). As the name suggests, these gels can be reliquified by heating to 65 °C and thus, for example, DNA samples separated in a gel can be cut out of the gel, returned to solution and recovered.

Owing to the poor elasticity of agarose gels and the consequent problems of removing them from small tubes, the gel rod system sometimes employed for acrylamide gels is not used. Horizontal slab gels are invariably used for isoelectric focussing or immunoelectrophoresis in agarose. Horizontal gels are also used routinely for DNA and RNA gels, although vertical systems have been used by some workers.

Different buffers can be used and are selected depending on the samples to be ana lysed. The most commonly used buffer for separating DNA is TRIS-acetate containing EDTA ( TAE) with a typical pH of 8.3 and used to dissolve the matrix (agarose) as well as the running buffer. This buffer suits the separation of dsDNA and allows rapid run times, even though it is a weak buffer and can warm considerably. For separation of RNA, TRIS-borate with EDTA ( TBE) is typically used due to superior buffering capacity (beneficial for long run times). However, borate acts as an inhibitor for many enzymes, which is problematic for downstream enzymatic steps such as ligation reactions in molecular biology. TBE gives superior separation of smaller fragments (< 2 kb) in comparison to TAE and is often used for separation of small RNA molecules such as microRNA.

Polyacrylamide Gels

Electrophoresis in acrylamide gels is frequently referred to as PAGE, being an abbreviation for PolyAcrylamide Gel Electrophoresis. Cross-linked polyacrylamide gels are formed from the polymerisation of acrylamide monomer in the presence of smaller amounts of N,N′ -methylene-bis-acrylamide (normally referred to as ‘ bis-acrylamide’) ( Figure 2). Note that bis-acrylamide is essentially two acrylamide molecules linked by a methylene group, and is used as a cross-linking agent. Acrylamide monomer is polymerised in a head-to-tail fashion into long chains and occasionally a bis-acrylamide molecule is built into the growing chain, thus introducing a second site for chain extension. Proceeding in this way, a cross-linked matrix of fairly well-defined structure is formed ( Figure 2). The polymerisation of acrylamide is an example of free-radical catalysis, and is initiated by the addition of ammonium persulfate and the base N,N,N′,N′ -tetramethylenediamine ( TEMED). TEMED catalyses the decomposition of the persulfate ion to give a free radical (i.e. a molecule with an unpaired electron):

If this free radical is represented as R • (where the dot represents an unpaired electron) and M as an acrylamide monomer molecule, then the polymerisation proceeds as follows:

Free radicals are highly reactive species due to the presence of an unpaired electron that needs to be paired with another electron to stabilise the molecule. R • therefore reacts with M, forming a single bond by sharing its unpaired electron with one from the outer shell of the monomer molecule. This therefore produces a new free radical molecule R−M •, which is equally reactive and will attack a further monomer molecule. In this way long chains of acrylamide are built up, being cross-linked by the introduction of the occasional bis-acrylamide molecule into the growing chain. Oxygen reacts with remaining free radicals and therefore all gel solutions are normally degassed (the solutions are briefly placed under vacuum to remove loosely dissolved air) prior to use. The degassing of the gel solution also serves a second purpose. The polymerisation of acrylamide is an exothermic reaction (i.e. heat is liberated) and the warming up of the gel solution as it sets can liberate air bubbles that become trapped in the polymerised gel. The degassing step prevents this possibility.

Fig2. The formation of a polyacrylamide gel network from acrylamide and bis-acrylamide.

Photopolymerisation is an alternative method that can be used to polymerise acryl amide gels. Ammonium persulfate and TEMED are replaced by riboflavin and when the gel is poured it is placed in front of a bright light for 2–3 h. Photodecomposition of riboflavin generates a free radical that initiates polymerisation.

Acrylamide gels are defined in terms of the total percentage of acrylamide present, and the pore size in the gel can be varied by changing the concentrations of both acrylamide and bis-acrylamide. Acrylamide gels can be made with a content of between 3% and 30% acrylamide; low-percentage gels (e.g. 4%) have large pore sizes and are used, for example, in the electrophoresis of proteins, where free movement of the proteins by electrophoresis is required without any noticeable frictional effect, for example in flat-bed isoelectric focussing or the stacking gel system of an SDS–polyacrylamide gel. Low-percentage acrylamide gels are also used to separate DNA. Gels of between 10 and 20% acrylamide are used in techniques such as SDS–gel electrophoresis, where the smaller pore size now introduces a sieving effect that contributes to the separation of proteins according to their size.

Proteins were originally separated on polyacrylamide gels that were polymerised in glass tubes, approximately 7 mm in diameter and about 10 cm in length. The tubes were easy to load and run, with minimum apparatus requirements. However, only one sample could be run per tube and, because conditions of separation could vary from tube to tube, comparison between different samples was not always accurate. The later introduction of vertical gel slabs allowed up to 20 samples to be run under identical conditions at the same time. Vertical slabs are used routinely both for the analysis of proteins and for the separation of DNA fragments during DNA sequence analysis. Although some workers prepare their own acrylamide gels, others purchase commercially available ready-made gels for techniques such as SDS–PAGE, native gels and isoelectric focussing (IEF).

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الأسباب الرئيسية لنقص الطاقة في الجسم

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الآخبار التكنلوجية

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جيتكس إفريقيا.. المغرب يجمع "رواد التكنولوجيا" من 103 دول

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المزيد

مواضيع ذات صلة

Principles of Liquid Chromatography

Preliminary Purification Steps

Cell Disruption and Production of Initial Crude Extract

Determination of Protein Concentrations

Molecular Biotechnology and Applications

The Biology of Restriction Enzymes

The Isolation of DNA

MODERN BIOTECHNOLOGY

ANCIENT BIOTECHNOLOGY

DEFINITIONS OF BIOTECHNOLOGY

Future Perspectives of Biofuels and Biotechnology

Biobutanol