electrophoresis technique

8/15/2019 Electrophoresis Technique

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WELCOME


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DATE:06.02.2014

ELECTROPHORESIS

TECHNIQUES

FACILIATED TO:DR. NAGRAJ GOWDA

H.O.D.

DEPT. OF PHARMACEUTICAL ANALYSISPESCP, BANGALORE

PRESENTED BY: MISS SAYANTI SAU I M. PHARM DEPT. OF PHARMACOLOGY PESCP, BANGALORE


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TYPES OF ELECTROPHORESIS

1) 'o"e Electrophoresis a) Paper Electrophoresis b) Gel Electrophoresis

c) Thin ayer Electrophoresis

d) !ellulose acetate Electrophoresis

") (o)i"# *o$" ary Electrophoresis a) !apillary Electrophoresis b) Isotachophoresis

c) Isoelectric #ocussing

d) Immuno Electrophoresis 4


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'ONE ELECTROPHORESISIt involves the m igration of the charged particle on the supporting media.

Paper, Cellulose acet ate m embrane, Starch Gel, Poly acrylamide.

Components s eparated are d istributed into discrete zon e on the su pport media.Supporting m edia is s aturated with buffer s olution, small volume of the sam ple is ap plied

as narrow band.

On application of PD at the en ds of a strip components m igrates at a rate determined by

its e lectrop horetic m obility.

ADVANTAGES:

Useful in biochemical investigations.

Small quanity of sample can be an alysed.

Cost is low and easy m aintenance .

DISADVANTAGES:

Unsuitable f or a ccurate m obility and isoelectric p oint determination.

Due t o the presence of supporting m edium, technical complications such as capillary ow,

electro o smosis, adsorption and molecular si eving are i ntroduced.

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+ENER,L (ETHOD OF OPER,TION

Saturation of the medium with the buffer.

Sample application.

Electrophoretic separation.

Removal of the supporting m edia.

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INSTRU(ENT,TIO N

Electrophoretic chamber.Electrodes.Diffusion barriers.Supporting/ Stabilizing media.

(inert to sample an d to an y developing reagents).


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P,PER ELECTROPHORESIS

1.Horizontal paper E lectrophoresis

2.Vertical paper Electrophoresis 7


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HORI'ONT,L P,PER ELECTROPHORESIS

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-ERTIC,L P,PER ELECTROPHORESIS


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Filter paper such as Whatmann no1 and no 3mm in strip of 3 or 5cm

wide h ave been used to good eff ect.

Separation takes p lace i n 12 to 14hrs.

ADVANTAGES:

It is econ omical.

Easy t o use.

DISADVANTAGES:

Certain compounds such as proteins, hydrophilic molecules cannot beresolved due t o t he a dsorptive a nd ionogenic properties of paper which

results i n tailing a nd distortion of component bands.

Electro osm osis.9


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CONTINUOUS ELECTROPHORESIS

• $sed for preparative scale purpose.•

Predetermined sample volume of %." ml&min through the valve device is appliedcontinuously on the centre of the paper.• 'ilica gel( powdered glass( sand agar gel can be used.• * P+ of ,%%- is applied.• Pure compounds are collected in separate containers( the solvent is evaporated and pure

fractions are reused.•

*fter complete electrophoresis the filter is removed from apparatus and stained to locatethe separated components.

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+EL ELECTROPHORESISSeparation is brought about through molecular sieving technique, based on themolecular s ize of the su bstances. Gel material acts as a " molecular si eve”.

Gel is a colloid in a solid form (99% is w ater).

It is i mportant that the su pport media is el ectrically neutral.Different types of gels which can be used are; Agar and Agarose gel, Starch,Sephadex, Polyacrylamide gels.

A porous gel acts as a sobstructing the movement of macromolecules while allowing smallermolecules t o migrate freely .

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+EL ELECTROPHORESISGel Electrophoresis

is ca ried out in twomethods:

1.Vertical st arch gel

electrophoresi s

2.Horizontal starchgel electrophoresi s

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,+,R ,ND ,+,ROSE +EL

Agar is a mix

Agarose is a

Agarose is chemically basic disaccharide repeating units of 3,6-anhydro-L-galactose .

Agarose dissoltemperature i s l owered to a bout 40° C at w hich point it gels.

The p ore size m ay b e p redetermined by adjusting t he concentration of agarose i n the gel .

Agarose gels are frg

together by the formation of weak hydrogen and hydrophobic bonds.The pores of an agarose gel are l arge, agarose i s u sed t o sepa rate m acromolecules s uch asnucleic aci ds, large p roteins an d protein complexes .

ADVANTAGES:

Easy to p repare an d small concentration of agar i s r equired.Resolution is superior to that of lter paper.

Large q uantities of proteins c an be separated a nd recovered.

Adsorption of

It adsorbs p roteins relatively less when compared t o other medium.

Sharp zones are obt ained due t o less adsorption.Recovery of protein is good , good method for preparative p urpose.

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+EL STRUCTURE OF ,+,ROSE:

DISADVANTAGES:

Electro osm osis i s h igh.

Resolution is l ess c ompared to pol yacrylamide ge ls.

Different sources an d batches of agar tend to give d ifferent results an d purication is

often necessary.

APPLICATION:

Widely used in Immuno electrophoresis.

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It is prepared by polymerizing acryl amide monomers inthe presence of methylene-bis-acrylamide to cross linkthe monomers.• Structure of acrylamide (CH 2=CH-CO-NH 2)• Polyacrylamide gel structure h eld together b y covalent

cross-links.• Polyacrylamide gel s are t ougher t han agarose gel s.• It is thermostable, transparent, strong and relatively

chemically inert.• Gels are u ncharged and are prepared in a v ariety of pore s izes.• Proteins are separated on the basis of charge to mass

ratio and molecular size, a phenomenon calledMolecular si eving.

ADVANTAGES:Gels ar e stable over wide ran ge of pH and t emperature.

Gels of different pore s ize c an be formed.

Simple a nd separation speed is good comparatively.

POLY,CRYL,(IDE +EL ELECTROPHORESIS.P,+E/

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TYPES OF P,+EPAGE can be classied according the

separation conditions into :

NATIVE-PAGE:

Native gels are ru n in non-denaturing conditions, so

that the a nalyte's n atural structure i s m aintained.

Separation is based u poncharge, size, and shape

ofmacromolecules.

Useful for separation or purication of mixture of

proteins.

This w as t he ori ginal mode of electrophoresis .DENATURED-PAGE OR SDS-PAGE:

Separation is based u pon the molecular weight of

proteins.

The common method for determining MW of proteins.

Very useful for checking puri

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P,+E0PROCEDUREThe g el of different pore si zes i s cast into a column inside a vertical tube, often with

large p ore gel at the t op and small pore gel at the b ottom.

Microgram quantity of the sample is placed over the top of the gel column and

covered by a buffer solution having such a pH so as t o change sample components

into a nions.

The foot of the gel column is m ade t o d ip in the sam e bu ffer in the b ottom reservoir.

Cathode an d anode are kept above and below the column to impose an electric eld

through the column.

Macromolecular anions m ove towards t he an ode down the gel column.

There i s n o ex ternal solvent space, all the m igratory p articles h ave t o pass through

the g el pores.

Rate of migration depends on the charge t o mass ratio.

Different sample components get separated into discrete m igratory ba nds along the

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Polyacrylamide Gel ElectrophoresisP*GE)

a) The gel is poured vertically between two glass plates. b.) Protein bands are separated on the basis of relative molecular weight andvisuali/ed with stains.

SLAB PAGEP,+E PROCEDURE

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SL,* P,+EThe P olyacrylamide g el is cast as t hin rectangular sl ab inside a plastic frame a nd this

slab is p laced vertically on a buffer sol ution taken in a reservoi r.

Several samples d issolved in dense su crose sol ution or gl ycerol are p laced in separate

wells cut in to the upper edg e of the sl ab and are co vered b y the sam e buffer s olution.

Cathode and anode are ab ove and below to produce electric eld effect. Different

components migrate simultaneously down parallel lanes in the slab and get separat ed

into ba nds.

VISUALIZATION

After the ele

them visible.

Ethidium bromide, silver, or coo massie blue dye may b e u sed for t his process .

If the a nalyte m olecules uoresce under ultra violet light, a p hotograph can be t aken of

the gel under u ltraviolet lighting conditions. If the m olecules t o be sep arated contain

radioactivity added for v isibility, an autoradiogram can be rec orded of the ge l.19

SDS0POLYCRYL (IDE EL


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SDS0POLY,CRYL,(IDE +ELELECTROPHORESIS .SDS0P,+E /

SDS-PAGE , sodium dodecyl sulfate polyacrylamide gel electrophoresis , is a

technique widely used in biochemistry, forensics, genetics and molecular biology to

separate p roteins a ccording to t heir electrophoretic mobility.

0hen a detergent '+' added to P*GE the combined procedure is termed as '+'

P*GE.

SDS coats protei" molec$les #i)i"# all protei"s a co"sta"t char#e0mass ratio&

D$e to mas1i"# o! char#es o! protei"s by the lar#e "e#ati)e char#e o" SDS bi" i"#

2ith them3 the protei"s mi#rate alo"# the #el i" or er o! i"creasi"# si4es or

molec$lar 2ei#hts&

SDS is an anionic detergent which denatures secondary and non–disulde–linked

tertiary st ructures by w rapping around the polypeptide backbone. In so doing, SDS

confers a n et negative ch arge t o t he p olypeptide i n proportion to i ts l ength.

Molecules i n solution with SDS have a net negative charge w ithin a wide pH range.

A polypeptide chain bn

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SODIU( DODECYL SULF,TE .SDS0P,+E/

Native protein is unfolded by heating in the

presence of -mercaptoethanol and SDS.

SDS binds to the protein so that it stays in

solution and denatures.Large polypeptides bind more SDS than

small polypeptides, so proteins end up

with negative charge in relation to their

size.

When treated with SDS and a reducing

agent, the polypeptides become rods of

negative charges with equal “charge

densities" or c harge p er u nit length.

Thus, we can separate the proteins based on

Native p rotein

Heat+

Reductant+

SDS

Denatured proteinwith bound SDS

N

C

- -

-

---

- -

- -

--

- --

--

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ST,RCH A suspension of

clear col loidal suspension.

The suspension on cooling sets as a semisolid gel due tointertwining of the b ranched chains of amylopectin.In order t o av oid swelling a nd shrinking p etroleum jelly i s u sed.

ADVANTAGES:o High resolving power and sharp zones are o btained.o The components resolved can be recovered in reasonable yield

especially proteins.o Can be u sed for a nalytical as w ell as p reparative electrophoresis.

DIS,D-,NT,+ES:o Electro osm otic effect.o Variation i 23


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-ERTIC,L ,ND HORI'ONT,L+EL ELECTROPHORESIS

Methods for application

of sample:

a) Sample is mixed with

the g el.

b) By soaking a bi t of lter

paper in sample and

pressi ng it into th e g el.

c) Detection by staining or

by Direct gravimetry or

by UV absorption, etc.

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Pulsed eld gel electrophoresis is a t echnique used for the separation of large

DNA molecules by applying to a gel matrix an electric eld that periodically

changes d irection.

While in general small fragments can nd their way through the gel matrix more easi ly

than large D NA fragments, a threshold length exists above 3 0–50 kb where all large

fragments will run at the sam e rate, and appear in a gel as a si ngle large d iffuse ba nd.

However, with periodic changing of eld direction, the various lengths of DNA

react t o the change at differing rates. Over t he course of time with the consistent

changing of directions, each band will begin to sepa rate m ore an d more e ven at ver y large

lengths. Thus s eparation of very large D NA pieces usi ng P FGE is m ade p ossible.

The voltage is periodically switched among three directions; one that runs

through the central axis of the gel and two that run at an angle of 60 degreeseither side. The pulse times are e qual for each direction resulting in a net

forward migration of the DNA.

This proced ure takes longer than normal gel electrophoresis due to the size of the

fragments being resolved and the fact that the DNA does not move in a straight line

through the gel.

PULSED0FIELD +EL ELECTROPHORESIS


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,PPLIC,TIONS:

PFGE may be u sed for genotyping or gen etic ngerprinting . It is commonly

considered a gold standard in epidemiological studies of pathogenic or ganisms.


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THIN L,YER ELECTROPHORESIS

Studies ca n be ca rried out in thin layer of silica, keisulguhr, alumina.

ADVANTAGES:Less time consuming a nd good resolution .

APPLICATION:

Widely used in combined electrophoretic-chromatography studies in

two d imensional study of proteins an d nucleic acid hydrolysates.

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CELLULOSE ,CET,TEELECTROPHORESISIt contains 2 -3 acetyl groups p er gl ucose u nit and its a dsorption capacity is lessthan that of paper.

It gives s harper ba nds.Provides a good background for s taining glycoproteins .

ADVANTAGE:No tai ling of proteins or h ydrophilic m aterials.

Available in

Give sharp b ands and offer good res olution.

High voltage can be applied which will enhance t he res olution.

DISADVANTAGE:Expensive.

Presence of sulphonic and carboxylic residue causes induced electroosmosisduring el ectrophoresis.

APPLICATION:Widely used in analysis of clinical and biological protein samples

(albumin and globulins).

Alternative

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(O IN *OUND RY ELECTROPHORESIS


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(O-IN+ *OUND,RY ELECTROPHORESISPRINCIPLE:The moving boundary method allows the charged species to migrate in a freemoving solution without the supporting medium.

INSTRUMENTATION:o Consists of a U shaped glass c ell of rectangular c ross s ection, with electrodes p laced on

the on e ea ch o f the l imbs of the cel l.o Sample solution is introduced at the bottom or through the side arm, and the

apparatus i s pl aced in a constant temp. bath at 40 o C.o Detection is done by measuring ref ractive index throughout the solution.(Schlieren

optical system ).

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,D-,NT,+ES:

Biologically active fractions c an be recovered without the u se of denaturing

agents. A reference method

Minute concentrations of the sample can be detected.(0.05mg/ml by

Interferometric optical system).

DIS,D-,NT,+ES:

Costlier.

Elaborate op tical system are requ ired.

,PPLIC,TION:

To study h omogenecity of a m acromolecular s ystem.

Analysis of p

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C,PILL,RY ELECTROPHORESIS• The principle behind electrophoresis is that charged molecules will migrate toward the

opposite p ole a nd separat e from each other based on physical characteristics .• Capillary electrophoresis has grown to become a collection of a range of separation

techniques w hich involve the a pplication of high voltages a cross b uffer lled capillaries t o

achieve separations .• Capillary electrophoresis, then, is the t echnique of performing electrophoresis in

buffer-lled, narrow-bore capillaries , normally from 25 to 100 mm in internal

diameter (ID).• A high voltage (• Capillaries are t ypically of 50 µm inner di ameter an d 0.5 to 1 m in length .• Due t o el ectroosmotic ow, all sample com ponents m igrate t owards t he n egative el ectrode.• The capillary can also be lled with a gel, which eliminates the electroosmotic ow.

Separation is accom plished as in conventional gel electrophoresis bu t the cap illary a llowshigher reso lution, greater se nsitivity, and on-line d etect ion.

• The ca pillary i s lled with electrolyte sol ution which conducts cu rren t through the inside of

the ca pillary. The e nds of the ca pillary a re d ipped into rese rvoirs lled with the e lectrolyte.• Electrodes ( platinum) are i nsert ed into the e lectrolyte rese rvoirs t o com plete t he e lectrical

circu it.

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Sample a pplication is d one b y eithera)High voltage injection -potential is a pplied causing the sa mple t o enter cap illary b y

combination of ionic at traction and electroosmotic ow.

b)Pressure injection -pressure d ifference i s u sed to drive the sa mple into ca pillary b yapplying vaccum.• When PD is ap plied net migration occurs in the d irection of cathode.• Even substance with net negative charge m igrate in the d irection of cathode d ue t o the

phenomenon cal led as E lectro Osmotic Flow.• Neutral molecule m oves at the same speed a s the E OF. Positively charged speci es move

faster, speed is s um of EOF and Electrophoretic mobility. Negatively ch arged moleculeslag b ehind .


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ELECTROOS(OTIC FLO5

The surface of the silicate glass capillary contains negatively-charged

functional groups that attract positively-charged counterions. The positively-

charged ions migrate towards the negative electrode and carry solvent

molecules in the sam e direction. This overall solvent movement is called

electroosmotic ow. During a separation, uncharged molecules move at the

same velocity as the electroosmotic ow (with very little separation).

Positively-charged ions m ove f aster a nd negatively-charged ions m ove sl ower.

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• A small volume of sample ithrough a detector, usually a UV absorbance det ector, at the opposite end of thecapillary.

• Application of a voelectrode usually passing through the detector.

• A plot of detector response with time is generated which is termed anelectropherogram.

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We ll one well with slowtrailing electrolyte (T) mixedwith samples (S1,S2), and theother well with fast leadingelectrolyte (L). When we applyan electri c eld, ions

electromigrate through amicrochannel according to theirelectrop horeti c mobilities .

Sample ions overspeed t he slowtrailing electrol yte, but cannotoverspeed the fast leadingelectrol yte ; consequently, they

focus a t the interface .

Sample continues toaccumulate. If sampleconcentration approachesthe concentration of the l eadingelectrol yte, samples self-segregate i nto d iscrete zon es.

The a nalytes are p ositioned between the el ectrolytes and, when the vol tage i s ap plied, they m igrate in


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order of decreasi ng mobility.

This est ablishes t he p otential gradient; from that point on, all the a nalytes m ove a t the sa me sp eed.

Individual zones border one a nother bu t represent completely sepa rated components w ith out overlap.

In isotachophoresis no background electrolyte(buffer) is mixed with the sam ple, so current ow is

carried only by ch arged sample i ons.

Once a f aster m oving component separates completely from a slower m oving one, It creates

a region of depleted charge between the two that increases the resistance and therefore

local voltage in that region.This increased vo ltage causes the slower component to migrate faster and close the gap,

thereby concentrating it and increasing the co nductivity of its zon e u ntil it matches t hat of

the f aster i on.

Ultimately all ions migration at the rate of the faster ion in the zon es that differ in thickness,

depending on their ori ginal concentrations.

APPLICATION:

Isotachophoresis t hat been used for t he sep aration of proteins a s w ell as

inorganic substances.

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I((UNO ELECTROPHORESIS


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I((UNO ELECTROPHORESIS Antibodies are produced une

binds spe cically to one feature(epitope) on one macromolecule(antigen).This allows the use of

antibodies f or d etection and quantitation of specic prot eins i n a complex m ixture.

When Electrical potential is ap plied to study of antigen-antibody reactions, it is ca lled

Immunoelectrophoresis. The antibody are electrophoretically separated and antigens

diffuse towards each other resulting in precipitin arcs where antigen antibody

complexes form. This technique has been referred to as i mmunoelectrophoresis.

Antibody is placed

Run the electrophoresis as a result precipitin arcs will be formed due to Ab-Ag complex

formation.

A uid containing prote

electric cu rren t is a pplied, antigens will be distributed in separate sp ots a long a line passing

through the w ell and parallel to t he d irection of current ow.

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(ETHOD


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(ETHODIt is u sually carr ied out in 2% agar gel medium.

The a ntigen mixture i s ap plied into a small circular w ells cut out of agar an d the initial

electrophoretic seperation is carrried out depending on their charge an d molecular weight.

After the

the gel about 0.5 to 1. 0 cm from the sep arated antigens.

During this peri od , the antigen components diffuse radially outwards, towards the

diffusing a ntibody and precipitation takes p lace i n elliptical arcs a s rel ated antigens a nd

antibodies d iffuse i nto on e a nother.

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,D-,NT,+ES:• Spreading of bands is m inimized d ue to the application of the ap plied eld and the pH

gradient, high resolution can be a chieved.

DIS,D-,NT,+ES:• Carrier a mpholytes ge nerally are u sed in relatively high concentration , a high voltage

power source ( up to 2000V) is necessary a nd power i s in the vicinity of 2 to 50 W.As aresult the e lectrophoretic m atrix must be cool ed.

,PPLIC,TION:• Mainly u sed f or s eparating p rotein and peptides.• Used in clinical, forensic and human genetics laboratories for the separation and

identication of serum protein in reseach in enzymology, membrane biochemistry,microbiology and immunology .

ISOELECTRIC FOCSIN+


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ISOELECTRIC FOCSIN+PRINCIPLE:

All proteins have an

When electrophoresis is r un in a sol ution buffered a t constant pH , proteins having a net charge

will migrate t owards t he op posite el ectrode so l ong as t he cu rrent ows.

The u se of pH gradient across the sup porting m edium causes each p rotein to m igrate t o an area ofspecic pH.The pH of the protein equals the pH of the grad ient, thus resulting in sharp welldened p rotein bands.

A procedure to determine the isoelectric poiproteins can be electrophorised through a solution having a stable pH gradient infrom the anode to the cathode and a each protein will m igrate to the position in thepH gradient a ccording to its isoelectric point. This is called isoelectric focusing.

Protein migrate i nto the p oint where i ts n et charge is zer o – isoelectric p H.

Protein is posi tively ch arged in solutions at pH below its P I and will migrate t owards t he cat hode.

Protein is n egatively charged in solution at pH above i ts P I w ill migrate t owards t he a node.

They w ill be i n the Zwitter ion form with no net charge so t he further movement will cease.

Ampholytes (amphoteric electrolytes)- low molecular mass (600-900D) ooligomers with aliphaticamino and carboxylic acid groups with a range of isoelectric points. Ampholytes help maintainthe pH gradiennt in the presence of high voltage.

Can also use gels with immobilized pH gradients - made of acrylamide derivatives that are

covalently linked to ampholytes.

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(ETHOD


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Ampholytespolyacrylamide

Cathode(-)electrodesolution

Anode(+)electrode

(ETHOD• pH gradient is established in gel by ad dition of ampholytes which increases the pH from anode t o cathode.• A protein mixture is pl• With an applied electric eld, proteins en ter t he g el migrates u ntil each reach es i ts p H equivalent to i ts ( PI).• Each species of proteins is therby f ocussed i nto a n arrow band a bout its PI.• The Anode of the column is connected to a reservoir containing an acidic solution like phosphoric

acid and Cathode is con nected to a reservoir con taining a lkaline solution like sod ium hydroxide.• On opening the two reservoir valves the two solutions are allowed to diffuse into the column from

their r espective en ds , setting u p a P H gradient between the acidic an ode an d the alkaline cat hode.• The va lves are t hen closed a nd the cu rrent is sw itched on , causing the carr ier am pholytes to migrate un til they

reach the P H regions where they h ave n o net charge. They w ill then remain stationary at t hese poi nts.

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T5O0DI(ENSION,L +EL ELECTROPHORESIS


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T5O0DI(ENSION,L EL ELECTROPHORESIS.60D ELECTROPHORESIS /

In the rst dimension, proteins are resol ved in according to their isoelectric

points (PI) using immobilized pH gradient electrophoresis (IPGE),

isoelectric focusing (IEF ), or n on-equilibrium pH gradient electrophoresis.

(Horizontal seperation)

In the second dimension, proteins are separated according to their

approximate molecular w eight using SDS-PAGE. (Vertical seperation ).

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PPLIC TIONS OF ELECTROPHORESIS


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,PPLIC,TIONS OF ELECTROPHORESIS1. DNA Sequencing

2. Medical Research

3. Protein research/purication

4. Agricultural testing5. Separation of organic acid, alkaloids, carbohydrates, amino acids, alcohols, phenols, nucleic

acids, insulin.

6. In food industry

7. It is employed in biochemical and clinical elds i.e. in the study of protein mixtures such as

blood serum, haemoglobins an d in the st udy of antigen- antibody i nteractions.8. Electrophoresis in combination with autoradiography is used t o study the binding of iron t o

serum proteins.

9. Used for a nalysis of terpenoids , steroids a nd antibiotics.

10.For testing purity of thyroid h ormones by zon e el ectrophoresis.

11.Paper c hromato-electrophoresis i s u sed to sep arate f ree I nsulin from plasma proteins.12.It is u sed for d iagnosis of various d iseases of kidney , liver a nd CVS.

13.It is al so used for s eparation of Scopolamine an d Ephedrine u sing b uffer at PH 4.2.

14.Electrophoresis i s al so u sed for s eparation of carbohydrates an d vitamins.

15.Quantitative separati on of all fracti ons of cellular en tities, antibiotics, RBC, Enzymes etc is

possible.

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electrophoresis technique

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