Gel electrophoresis

Course: B.Sc Biotechnology

Subject: Bio Analytical Technique

Unit: III

Gel electrophoresis

• Gel electrophoresis is a method for separation and analysis of macromolecules(DNA, RNA and proteins) and their fragments, based on their size and charge.

• It is used in clinical chemistry to separate proteins by charge and/or size and inbiochemistry and molecular biology to separate a mixed population of DNA andRNA fragments by length, to estimate the size of DNA and RNA fragments or toseparate proteins by charge.

• The term electrophoresis describes the migration of a charged particle under theinfluence of an electric field.

• Many important biological molecules, such as amino acids, peptides, proteins,nucleotides and nucleic acids, possess ionisable groups and, therefore, at anygiven pH, exist in solution as electrically charged species either as cations (+) oranions (-).

• Under the influence of an electric field these charged particles will migrate eitherto the cathode or to the anode, depending on the nature of their net charge.

• Nucleic acid molecules are separated by applying an electric field to move thenegatively charged molecules through a matrix of agarose or other substances.

• Shorter molecules move faster and migrate farther than longer ones becauseshorter molecules migrate more easily through the pores of the gel. Thisphenomenon is called sieving.

• Proteins are separated by charge in agarose because the pores of the gel are toolarge to sieve proteins.

Why Gel is use as a Medium..?

• Gel electrophoresis uses a gel as an anticonvective medium and/orsieving medium during electrophoresis, the movement of a chargedparticle in an electrical field.

• Gels suppress the thermal convection caused by application of theelectric field, and can also act as a sieving medium, retarding thepassage of molecules.

• Gels can also simply serve to maintain the finished separation, sothat a post electrophoresis stain can be applied.

• DNA Gel electrophoresis is usually performed for analyticalpurposes, often after amplification of DNA via PCR, but may beused as a preparative technique prior to use of other methods suchas mass spectrometry, RFLP, PCR, cloning, DNA sequencing, orSouthern blotting for further characterization.

Types of gel

• The types of gel most typically used are agarose and polyacrylamidegels.

• Each type of gel is well-suited to different types and sizes ofanalyte.

• Polyacrylamide gels are usually used for proteins, and have veryhigh resolving power for small fragments of DNA (5-500 bp).

• Agarose gels on the other hand have lower resolving power forDNA but have greater range of separation, and are therefore usedfor DNA fragments of usually 50-20,000 bp in size, but resolution ofover 6 Mb is possible with pulsed field gel electrophoresis (PFGE).

• Polyacrylamide gels are run in a vertical configuration while agarosegels are typically run horizontally in a submarine mode.

• They also differ in their casting methodology, as agarose setsthermally, while polyacrylamide forms in a chemical polymerizationreaction.

Agarose

• Agarose gels are made from the natural polysaccharide polymers extracted fromseaweed.

• Agarose gels are easily cast and handled compared to other matrices, because thegel setting is a physical rather than chemical change.

• Samples are also easily recovered.• After the experiment is finished, the resulting gel can be stored in a plastic bag in a

refrigerator.• Agarose gels do not have a uniform pore size, but are optimal for electrophoresis

of proteins that are larger than 200 kDa.• Agarose gel electrophoresis can also be used for the separation of DNA fragments

ranging from 50 base pair to several megabases (millions of bases), the largest ofwhich require specialized apparatus.

• The distance between DNA bands of different lengths is influenced by the percentagarose in the gel, with higher percentages requiring longer run times, sometimesdays.

• "Most agarose gels are made with between 0.7% and 2% agarose dissolved inelectrophoresis buffer.

• Low percentage gels are very weak and may break when you try to lift them. Highpercentage gels are often brittle and do not set evenly.

• 1% gels are common for many applications.

Polyacrylamide

• Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins rangingin size from 5 to 2,000 kDa due to the uniform pore size provided by thepolyacrylamide gel.

• Pore size is controlled by modulating the concentrations of acrylamide and bis-acrylamide powder used in creating a gel.

• Care must be used when creating this type of gel, as acrylamide is a potentneurotoxin in its liquid and powdered forms.

• Traditional DNA sequencing techniques such as Maxam-Gilbert or Sanger methodsused polyacrylamide gels to separate DNA fragments differing by a single base-pairin length so the sequence could be read.

• Most modern DNA separation methods now use agarose gels, except forparticularly small DNA fragments.

• It is currently most often used in the field of immunology and protein analysis,often used to separate different proteins or isoforms of the same protein intoseparate bands. These can be transferred onto a nitrocellulose or PVDF membraneto be probed with antibodies and corresponding markers, such as in a westernblot.

• Typically resolving gels are made in 6%, 8%, 10%, 12% or 15%. Stacking gel(5%) is poured on top of the resolving gel and a gel comb is inserted.

• The percentage chosen depends on the size of the protein that one wishesto identify or probe in the sample.

• The smaller the known weight, the higher the percentage that should beused. Changes on the buffer system of the gel can help to further resolveproteins of very small sizes.

Starch

• Partially hydrolysed potato starch makes for another non-toxic medium forprotein electrophoresis.

• The gels are slightly more opaque than acrylamide or agarose.• Non-denatured proteins can be separated according to charge and size.• They are visualised using Napthal Black or Amido Black staining. Typical

starch gel concentrations are 5% to 10%.

Buffers

• Buffers in gel electrophoresis are used to provide ions that carry acurrent and to maintain the pH at a relatively constant value. Thereare a number of buffers used for electrophoresis. The mostcommon being, for nucleic acids Tris/Acetate/EDTA (TAE).

• In most cases the purported rationale is lower current (less heat)and or matched ion mobilities, which leads to longer buffer life.

• Most SDS-PAGE protein separations are performed using a"discontinuous" (or DISC) buffer system that significantly enhancesthe sharpness of the bands within the gel. During electrophoresis ina discontinuous gel system, an ion gradient is formed in the earlystage of electrophoresis that causes all of the proteins to focus intoa single sharp band in a process called isotachophoresis.

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Applications

• Estimation of the size of DNA molecules followingrestriction enzyme digestion, e.g. in restrictionmapping of cloned DNA.

• Analysis of PCR products, e.g. in moleculargenetic diagnosis or genetic fingerprinting

• Separation of restricted genomic DNA prior toSouthern transfer, or of RNA prior to Northerntransfer.

• Gel electrophoresis is used in forensics, molecularbiology, genetics, microbiology and biochemistry.

Agarose gel electrophoresis

• Agarose gel electrophoresis is a method of gel electrophoresis used inbiochemistry, molecular biology, and clinical chemistry to separate amixed population of DNA or proteins in a matrix of agarose.

• The proteins may be separated by charge and/or size, and the DNA andRNA fragments by length. Biomolecules are separated by applying anelectric field to move the charged molecules through an agarose matrix,and the biomolecules are separated by size in the agarose gel matrix.

• Agarose gels are easy to cast and are particularly suitable for separatinglarger DNA of size range most often encountered in laboratories, whichaccounts for the popularity of its use.

• The separated DNA may be viewed with stain, most commonly under UVlight, and the DNA fragments can be extracted from the gel with relativeease.

• Most agarose gels used are between 0.7 - 2% dissolved in a suitableelectrophoresis buffer.

Factors affect migration of nucleic acid in gel

• The dimension of the gel pores (gel concentration),

• Size of DNA being electrophoresed,

• The voltage used,

• The ionic strength of the buffer, and

• The concentration intercalating dye such as ethidium bromide if used during electrophoresis.

Mechanism of migration and separation

• The negative charge of its phosphate backbone moves the DNAtowards the positively-charged anode during electrophoresis.

• However, the migration of DNA molecules in solution, in theabsence of a gel matrix, is independent of molecular weight duringelectrophoresis.

• The gel matrix is therefore responsible for the separation of DNA bysize during electrophoresis, and a number of models exist to explainthe mechanism of separation of biomolecules in gel matrix.

• A globular protein or a random coil DNA moves through theinterconnected pores, and the movement of larger molecules ismore likely to be impeded and slowed down by collisions with thegel matrix, and the molecules of different sizes can therefore beseparated in this sieving process.

General procedure

1. Casting of gel• The gel is prepared by dissolving the agarose powder in an appropriate buffer,

such as TAE or TBE, to be used in electrophoresis.• The agarose is dispersed in the buffer before heating it to near-boiling point, but

avoid boiling.• The melted agarose is allowed to cool sufficiently before pouring the solution into

a cast as the cast may warp or crack if the agarose solution is too hot.• A comb is placed in the cast to create wells for loading sample, and the gel should

be completely set before use.• The concentration of gel affects the resolution of DNA separation.• For a standard agarose gel electrophoresis, a 0.8% gives good separation or

resolution of large 5–10kb DNA fragments, while 2% gel gives good resolution forsmall 0.2–1kb fragments. 1% gels are common for many applications.

• The concentration is measured in weight of agarose over volume of buffer used.High percentage gels are often brittle and may not set evenly, while lowpercentage gels (01.-0.2%) are fragile and not easy to handle.

2. Loading of samples• Once the gel has set, the comb is removed, leaving wells where

DNA samples can be loaded.• Loading buffer is mixed with the DNA sample before the mixture is

loaded into the wells.• The loading buffer contains a dense compound, which may be

glycerol, sucrose, or Ficoll, that raises the density of the sample sothat the DNA sample may sink to the bottom of the well.

• If the DNA sample contains residual ethanol after its preparation, itmay float out of the well.

• The loading buffer also include colored dyes such as xylene cyanoland bromophenol blue used to monitor the progress of theelectrophoresis.

• The DNA samples are loaded using a pipette.

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3. Electrophoresis

• Agarose gel electrophoresis is most commonly done horizontally in a submarinemode whereby the slab gel is completely submerged in buffer duringelectrophoresis.

• It is also possible, but less common, to perform the electrophoresis vertically, aswell as horizontally with the gel raised on agarose legs using the appropriateapparatus.

• The buffer used in the gel is the same as the running buffer in the electrophoresistank, which is why electrophoresis in the submarine mode is possible with agarosegel.

• For optimal resolution of DNA greater than 2 kb in size in standard gelelectrophoresis, 5 to 8 V/cm is recommended.

• Voltage may also be limited by the fact that it heats the gel and may cause the gelto melt if it is run at high voltage for a prolonged period, especially if the gel usedis LMP agarose gel.

• Too high a voltage may also reduce resolution, as well as causing band streakingfor large DNA molecules.

• Too low a voltage may lead to broadening of band for small DNA fragments due todispersion and diffusion.

• Since DNA is not visible in natural light, the progress of the electrophoresis ismonitored using colored dyes.

• A DNA marker is also run together for the estimation of the molecular weight ofthe DNA fragments.

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4. Staining and visualization• DNA as well as RNA are normally visualized by staining with

ethidium bromide, which intercalates into the major grooves of theDNA and fluoresces under UV light.

• The ethidium bromide may be added to the agarose solution beforeit gels, or the DNA gel may be stained later after electrophoresis.

• Destaining of the gel is not necessary but may produce betterimages.

5. Downstream procedures• The separated DNA bands are often used for further procedures,

and a DNA band may be cut out of the gel as a slice, dissolved andpurified. The gels may also be used for blotting techniques.

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Polyacrylamide gel electrophoresis

• Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used inbiochemistry, forensics, genetics, molecular biology and biotechnology to separatebiological macromolecules, usually proteins or nucleic acids, according to theirelectrophoretic 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 beadded to remove this structure and turn the molecule into an unstructured linearchain 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 linearizedproteins.

• This procedure is called SDS-PAGE. In most proteins, the binding of SDS to thepolypeptide chain imparts an even distribution of charge per unit mass, therebyresulting in a fractionation by approximate size during electrophoresis.

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Procedure

1. Sample preparation• Samples may be any material containing proteins or nucleic acids. These

may be biologically derived, for example from prokaryotic or eukaryoticcells, tissues, viruses, environmental samples, or purified proteins. In thecase of solid tissues or cells, these are often first broken downmechanically.

• The sample to analyze is optionally mixed with a chemical denaturant if sodesired, 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 toeach protein in proportion to its mass.

• Urea breaks the hydrogen bonds between the base pairs of the nucleicacid, causing the constituent strands to separate. Heating the samples toat least 60 °C further promotes denaturation.

• A tracking dye may be added to the solution. This typically has a higherelectrophoretic mobility than the analytes to allow the experimenter totrack the progress of the solution through the gel during theelectrophoretic run.

2. Preparing acrylamide gels• 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.• A source of free radicals and a stabilizer, such as ammonium persulfate and TEMED

are added to initiate polymerization.• The polymerization reaction creates a gel because of the added bisacrylamide,

which can form cross-links between two polyacrylamide 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.• 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 thecomb can be removed and the gel is ready for electrophoresis.

3. Electrophoresis• Various buffer systems are used in PAGE depending on the nature of the

sample and the experimental objective. The buffers used at the anode andcathode may be the same or different.

• An electric field is applied across the gel, causing the negatively chargedproteins or nucleic acids to migrate across the gel from the negativeelectrode (the cathode) towards the positive electrode (the anode).

• Depending on their size, each biomolecule moves differently through thegel 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 voltageapplied across the gel; migration occurs more quickly at higher voltages,but these results are typically less accurate than at those at lowervoltages.

• After the set amount of time, the biomolecules have migrated differentdistances based on their size. Smaller biomolecules travel farther downthe gel, while larger ones remain closer to the point of origin.

• Biomolecules may therefore be separated roughly according to size, whichdepends mainly on molecular weight under denaturing conditions, butalso depends on higher-order conformation under native conditions.

• However, certain glycoproteins behave anomalously on SDS gels.

4. Further processing• Following electrophoresis, the gel may be stained (for

proteins, most commonly with Coomassie Brilliant Blue; fornucleic acids, ethidium bromide), allowing visualization ofthe separated proteins, or processed further (e.g. Westernblot).

• After staining, different species biomolecules appear asdistinct bands within the gel.

• It is common to run molecular weight size markers ofknown molecular weight in a separate lane in the gel tocalibrate the gel and determine the approximate molecularmass of unknown biomolecules by comparing the distancetraveled relative to the marker.

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Components use in PAGE1. Chemical buffer Stabilizes the pH value to the desired value within the gel itself and

in the electrophoresis buffer.• The choice of buffer also affects the electrophoretic mobility of the buffer

counterions and thereby the resolution of the gel.• The buffer should also be unreactive and not modify or react with most proteins.• Different buffers may be used as cathode and anode buffers, respectively,

depending on the application.• Multiple pH values may be used within a single gel, for example in DISC

electrophoresis. Common buffers in PAGE include Tris, Bis-Tris, or imidazole.

2. Acrylamide (C3H5NO; MW: 71.08). When dissolved in water, slow, spontaneousautopolymerization of acrylamide takes place, joining molecules together by headon tail fashion to form long single-chain polymers.

• The presence of a free radical-generating system greatly acceleratespolymerization.

• This kind of reaction is known as Vinyl addition polymerisation.• A solution of these polymer chains becomes viscous but does not form a gel,

because the chains simply slide over one another.• Gel formation requires linking various chains together.

3. Bisacrylamide (N,N'-Methylenebisacrylamide) (C7H10N2O2; mW: 154.17).• Bisacrylamide is the most frequently used cross linking agent for polyacrylamide

gels.• Chemically it can be thought of as two acrylamide molecules coupled head to head

at their non-reactive ends.• Bisacrylamide can crosslink two polyacrylamide chains to one another, thereby

resulting in a gel.

4. Sodium Dodecyl Sulfate (SDS) (C12H25NaO4S; mW: 288.38). (only used in denaturingprotein gels) SDS is a strong detergent agent used to denature native proteins tounfolded, individual polypeptides.

• When a protein mixture is heated to 100 °C in presence of SDS, the detergentwraps around the polypeptide backbone. It binds to polypeptides in a constantweight ratio of 1.4 g SDS/g of polypeptide.

• In this process, the intrinsic charges of polypeptides becomes negligible whencompared to the negative charges contributed by SDS.

• Thus polypeptides after treatment become rod-like structures possessing auniform charge density, that is same net negative charge per unit weight.

• The electrophoretic mobilities of these proteins is a linear function of thelogarithms of their molecular weights.

• Without SDS, different proteins with similar molecular weights would migratedifferently due to differences in mass-charge ratio. Adding SDS, it binds to andunfolds the protein, giving a near uniform negative charge along the length of thepolypeptide.

5. Urea (CO(NH2)2; mW: 60.06). (only used in denaturing nucleic acid gels) Urea is achaotropic agent that increases the entropy of the system by interfering withintramolecular interactions mediated by non-covalent forces such as hydrogenbonds and van der Waals forces.

• Macromolecular structure is dependent on the net effect of these forces,therefore it follows that an increase in chaotropic solutes denaturesmacromolecules.

6. Ammonium persulfate (APS) (N2H8S2O8; mW: 228.2). APS is a source of free radicalsand is often used as an initiator for gel formation.

• An alternative source of free radicals is riboflavin, which generated free radicals ina photochemical reaction.

• TEMED (N, N, N', N'-tetramethylethylenediamine) (C6H16N2; mW: 116.21). TEMEDstabilizes free radicals and improves polymerization.

• The rate of polymerisation and the properties of the resulting gel depend on theconcentrations of free radicals.

• Increasing the amount of free radicals results in a decrease in the average polymerchain length, an increase in gel turbidity and a decrease in gel elasticity.

• Decreasing the amount shows the reverse effect.• APS and TEMED are typically used at approximately equimolar concentrations in

the range of 1 to 10 mM.

Chemicals for processing and visualization

• Tracking dye. As proteins and nucleic acids are mostly colorless, theirprogress through the gel during electrophoresis cannot be easily followed.

• Anionic dyes of a known electrophoretic mobility are therefore usuallyincluded in the PAGE sample buffer. A very common tracking dye isBromophenol blue.

• This dye is coloured at alkali and neutral pH and is a small negativelycharged molecule that moves towards the anode.

• Being a highly mobile molecule it moves ahead of most proteins. As itreaches the anodic end of the electrophoresis medium electrophoresis isstopped.

• It can weakly bind to some proteins and impart a blue colour.

• Loading aids. Most PAGE systems are loaded from the top into wellswithin the gel. To ensure that the sample sinks to the bottom of the gel,sample buffer is supplemented with additives that increase the density ofthe sample. These additives should be non-ionic and non-reactive towardsproteins to avoid interfering with electrophoresis. Common additives areglycerol and sucrose.

3. Coomassie Brilliant Blue. CBB is the most popular protein stain. It is ananionic dye, which non-specifically binds to proteins.

• The structure of CBB is predominantly non-polar, and it is usually used inmethanolic solution acidified with acetic acid.

• Proteins in the gel are fixed by acetic acid and simultaneously stained.• The excess dye incorporated into the gel can be removed by destaining

with the same solution without the dye.• The proteins are detected as blue bands on a clear background.• As SDS is also anionic, it may interfere with staining process.• Therefore, large volume of staining solution is recommended, at least ten

times the volume of the gel.

4. Ethidium bromide (EtBr) is the traditionally most popular nucleic acidstain.

Native (buffer) gels

• While SDS–PAGE is the most frequently used gel system for studying proteins, the• method is of no use if one is aiming to detect a particular protein (often an

enzyme) on the basis of its biological activity, because the protein (enzyme) isdenatured by the SDS–PAGE procedure.

• In this case it is necessary to use non-denaturing conditions.• In native or buffer gels, polyacrylamide gels are again used (normally a 7.5% gel)

but the SDS is absent and the proteins are not denatured prior to loading.• Since all the proteins in the sample being analysed carry their native charge at the

pH of the gel (normally pH 8.7), proteins separate according to their differentelectrophoretic mobilities and the sieving effects of the gel.

• It is therefore not possible to predict the behaviour of a given protein in a buffergel but, because of the range of different charges and sizes of proteins in a givenprotein mixture, good resolution is achieved.

• The enzyme of interest can be identified by incubating the gel in an appropriatesubstrate solution such that a coloured product is produced at the site of theenzyme.

• An alternative method for enzyme detection is to includethe substrate in an agarose

• gel that is poured over the acrylamide gel and allowed toset.

• Diffusion and interaction of enzyme and substrate betweenthe two gels results in colour formation at the site of theenzyme.

• Often, duplicate samples will be run on a gel, the gel cut inhalf and one half stained for activity, the other for totalprotein.

• In this way the total protein content of the sample can beanalysed and the particular band corresponding to theenzyme identified by reference to the activity stain gel.

Gradient gels

• This is again a polyacrylamide gel system, but instead of running a slab gel ofuniform pore size throughout (e.g. a 15% gel) a gradient gel is formed, where theacrylamide concentration varies uniformly from, typically, 5% at the top of the gelto 25% acrylamide at the bottom of the gel.

• The gradient is formed via a gradient mixer and run down between the glass platesof a slab gel.

• The higher percentage acrylamide (e.g. 25%) is poured between the glass platesfirst and a continuous gradient of decreasing acrylamide concentration follows.

• Therefore at the top of the gel there is a large pore size (5% acrylamide) but as thesample moves down through the gel the acrylamide concentration slowlyincreases and the pore size correspondingly decreases.

• Gradient gels are normally run as SDS gels with a stacking gel.• There are two advantages to running gradient gels.• First, a much greater range of protein Mr values can be separated than on a fixed-

percentage gel.• In a complex mixture, very low molecular weight proteins travel freely through the

gel to begin with, and start to resolve when they reach the smaller pore sizestowards the lower part of the gel.

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• Much larger proteins, on the other hand, can still enter the gel butstart to separate immediately due to the sieving effect of the gel.

• The second advantage of gradient gels is that proteins with verysimilar Mr values may be resolved, although they cannot otherwisebe resolved in fixed percentage gels.

• As each protein moves through the gel the pore sizes becomesmaller until the protein reaches its pore size limit.

• The pore size in the gel is now too small to allow passage of theprotein, and the protein sample stacks up at

• this point as a sharp band.• A similar-sized protein but with slightly lower Mr will be able to

travel a little further through the gel before reaching its pore sizelimit, at which point it will form a sharp band.

• These two proteins, of slightly different Mr values, thereforeseparate as two, close, sharp bands.

Isoelectric focussing gels

• This method is ideal for the separation of amphoteric substances such asproteins because it is based on the separation of molecules according totheir different isoelectric points.

• The method has high resolution, being able to separate proteins that differin their isoelectric points by as little as 0.01 of a pH unit.

• The most widely used system for IEF utilises horizontal gels on glass platesor plastic sheets.

• Separation is achieved by applying a potential difference across a gel thatcontains a pH gradient. The pH gradient is formed by the introduction intothe gel of compounds known as ampholytes, which are complex mixturesof synthetic polyaminopolycarboxylic Acids.

• Ampholytes can be purchased in different pH ranges covering either awide band (e.g. pH 3-10) or various narrow bands (e.g. pH 7-8), and a pHrange is chosen such that the samples being separated will have theirisoelectric points (pI values) within this range.

• Commercially available ampholytes include Bio-Lyte and Pharmalyte.

• Traditionally 1-2mm thick IEF gels have been used by research workers,but the relatively high cost of ampholytes makes this a fairly expensiveprocedure if a number of gels are to be run.

• However, the introduction of thin-layer IEF gels, which are only 0.15mmthick and which are prepared using a layer of electrical insulation tape asthe spacer between the gel plates, has considerably reduced the cost ofpreparing IEF gels, and such gels are now commonly used.

• Since this method requires the proteins to move freely according to theircharge under the electric field, IEF is carried out in low percentage gels toavoid any sieving effect within the gel.

• Polyacrylamide gels (4%) are commonly used, but agarose is also used,especially for the study of high Mr proteins that may undergo somesieving even in a low percentage acrylamide gel.

• To prepare a thin-layer IEF gel, carrier ampholytes, covering a suitable pHrange, and riboflavin are mixed with the acrylamide solution, and themixture is then poured over a glass plate (typically 25 cm 10 cm), whichcontains the spacer.

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• The second glass plate is then placed on top of the first to form thegel cassette, and the gel polymerised by photopolymerisation byplacing the gel in front of a bright light.

• The photodecomposition of the riboflavin generates a free radical,which initiates polymerisation. This takes 2-3 h.

• Once the gel has set, the glass plates are prised apart to reveal thegel stuck to one of the glass sheets.

• Electrode wicks, which are thick (3 mm) strips of wetted filter paperare laid along the long length of each side of the gel and a potentialdifference applied.

• Under the effect of this potential difference, the ampholytes form apH gradient between the anode and cathode.

• The power is then turned off and samples applied by laying on thegel small squares of filter paper soaked in the sample.

• A voltage is again applied for about 30 min to allow the sample to electrophoreseoff the paper and into the gel, at which time the paper squares can be removedfrom the gel.

• Depending on which point on the pH gradient the sample has been loaded,proteins that are initially at a pH region below their isoelectric point will bepositively charged and will initially migrate towards the cathode.

• As they proceed, however, the surrounding pH will be steadily increasing, andtherefore the positive charge on the protein will decrease correspondingly untileventually the protein

• arrives at a point where the pH is equal to its isoelectric point.• The protein will now be in the zwitterion form with no net charge, so further

movement will cease.• Likewise, substances that are initially at pH regions above their isoelectric points

will be negatively charged and will migrate towards the anode until they reachtheir isoelectric points and become stationary.

• It can be seen that as the samples will always move towards their isoelectric pointsit is not critical where on the gel they are applied.

• To achieve rapid separations (2-3 h) relatively high voltages (up to 2500 V) areused.

• As considerable heat is produced, gels are run on cooling plates (10 C) and powerpacks used to stabilise the power output and thus to minimise thermalfluctuations.

• Following electrophoresis, the gel must be stained to detect theproteins.

• However, this cannot be done directly, because the ampholytes willstain too, giving a totally blue gel.

• The gel is therefore first washed with fixing solution (e.g. 10% (v/v)trichloroacetic acid).

• This precipitates the proteins in the gel and allows the muchsmaller ampholytes to be washed out. The gel is stained withCoomassie Brilliant Blue.

• The pI of a particular protein may be determined conveniently byrunning a mixture of proteins of known isoelectric point on thesame gel.

• A number of mixtures of proteins with differing pI values arecommercially available, covering the pH range 3.5-10.

• After staining, the distance of each band from one electrode ismeasured and a graph of distance for each protein against its pI(effectively the pH at that point) plotted.

• By means of this calibration line, the pI of an unknown protein canbe determined from its position on the gel.

• IEF is a highly sensitive analytical technique and is particularlyuseful for studying microheterogeneity in a protein.

• For example, a protein may show a single band on an SDS gel, butmay show three bands on an IEF gel.

• This may occur, for example, when a protein exists in mono-, di- andtri-phosphorylated forms.

• The difference of a couple of phosphate groups has no significanteffect on the overall relative molecular mass of the protein, hence asingle band on SDS gels, but the small charge difference introducedon each molecule can be detected by IEF.

• The method is particularly useful for separating isoenzymes, whichare different forms of the same enzyme often differing by only oneor two amino acid residues.

• Although IEF is used mainly for analytical separations, it can also beused for preparative purposes.

Two-dimensional polyacrylamide gel electrophoresis

• This technique combines the technique of IEF (first dimension), whichseparates proteins in a mixture according to charge (pI), with the sizeseparation technique of SDS–PAGE (second dimension).

• The combination of these two techniques to give two-dimensional (2-D)PAGE provides a highly sophisticated analytical method for analysingprotein mixtures.

• To maximise separation, most workers use large format 2-D gels (20 cm *20 cm), although the minigel system can be used to provide usefulseparation in some cases.

• For large-format gels, the first dimension (isoelectric focussing) is carriedout in an acrylamide gel that has been cast on a plastic strip (18 cm * 3mmwide).

• The gel contains ampholytes (for forming the pH gradient) together with8M urea and a non-ionic detergent, both of which denature and maintainthe solubility of the proteins being analysed.

• The denatured proteins therefore separate in this gel according to theirisoelectric points.

• The IEF strip is then incubated in a sample buffer containing SDS(thus binding SDS to the denatured proteins) and then placedbetween the glass plates of, and on top of, a previously prepared10% SDS–PAGE gel.

• Electrophoresis is commenced and the SDS-bound proteins run intothe gel and separate according to size.

• The IEF gels are provided as dried strips and need rehydratingovernight.

• The first dimension IEF run takes 6-8 h, the equilibration step withSDS sample buffer takes about 15 min, and then the SDS–PAGE steptakes about 5 h.

• Using this method one can routinely resolve between 1000 and3000 proteins from a cell or tissue extract and in some casesworkers have reported the separation of between 5000 and 10 000proteins.

Book and Web References

• Book Name : Wilson and Walker

• Book Name : Bioseparations: Principles and Techniques by B. Sivasankar

• http://cdn.intechopen.com/pdfs-wm/35088.pdf

• http://en.wikipedia.org/wiki/Gel_electrophoresis

• http://en.wikipedia.org/wiki/Polyacrylamide_gel_electrophoresis

• http://en.wikipedia.org/wiki/Agarose_gel_electrophoresis

Image References

• 1.http://upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE_Electrophoresis.png

• 2.http://www.mrcroce.com/uploads/1/4/0/1/14012789/4066539_orig.jpg?376

• 3.http://www.webbooks.com/MoBio/Free/images/Ch9C1.gif• Book Name: Principles and Techniques of Biochemistry and

Molecular Biology by Wilson n Walker• 4. http://media-3.web.britannica.com/eb-media/72/47672-004-

4E16B61F.jpg• 5.http://2009.igem.org/wiki/images/c/c1/KU_Seoul_4.jpg• 6. http://classroom.sdmesa.edu/eschmid/Lab12%201.jpg• 7. https://d15mj6e6qmt1na.cloudfront.net/i/2068425/600.jpg• 8.https://encryptedtbn0.gstatic.com/images?q=tbn:ANd9GcRPLSGt

xoPZYOzKe3suIGqlT3RLScxjUaA7ZZdHFw6Rb6jDi4WLRw

• 9.http://www.intechopen.com/source/html/38177/media/image2.png• 10.http://www.gibthai.com/userfiles/image/technote/Gel%20electrophor

esis/sds_page.png• 11.http://blog.universalmedicalinc.com/wpcontent/uploads/sites/136/gall

ery/postimages/dna-lab.jpg• 12.http://upload.wikimedia.org/wikipedia/en/thumb/6/68/Step-by

step_procedure_of_using_DGGE_analysis_in_microbiology.pdf/page1-1280px-Step-by-step_procedure_of_using_DGGE_analysis_in_microbiology.pdf.jpg

• 13.http://upload.wikimedia.org/wikipedia/commons/3/3d/Isoelectric_focusing_contribute2.jpg

• 14. http://www.aesociety.org/areas/images/ems_Fig1.jpg• 15.https://teamwork.jacobsuniversity.de:8443/confluence/download/atta

chments/4884460/pic05.jpg?version=1&modificationDate=1197407593000&api=v2