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Dr. D Y Patil Arts Commerce And Science College Pimpri Pune 411018 Chemistry Department Mrs. Tarannum S Attar CONTENT Introduction Principle Factors affecting Conventiona

l electrophoresis General operation Technical and practical Consideration Types of electrophoresis 3 I N T R O D U C TI O N Electrophoresis is the migration of charged pa

rticles or molecules in a medium under the influence of an applied electric field . 4 The rate of migration of an ion in electrical field depend on factors, o Net ch

arge of molecule o Size and shape of particle o Strength of electrical field o Properties of supporting medium o Temperature of operation o Ionic strength of buffer °

Peak broadening of samples ° Decrease resolution ° For m a t ion of t em per a ture gradi e nt – c on v e c t i on c urr e nt - resulting mixing of analytes ° Denaturatio

n of enzymes and proteins Effect of heat H=I 2 R Electro endosmosis C a thode Anode Conventional electrophoresis 18 B u ff e r The buffer in electrophoresis has twofold pu

rpose: Carry applied electrical current They set the pH at which electrophoresis is carried out. Thus they determine; Type of charge on solute. Extent of ionization of

solute Electrode towards which the solute will migrate. The buffer ionic strength will determine the thickness of the ionic cloud. 19 Commonly buffers used; B u ff e r 22 pH

value Phosphate buffer around 7.0 Tris - Borate - EDTA buffer (TBE) around 8.0 Tris - Acetate EDTAbuffer (TAE) above 8.0 Tris Glycine buffer (TG) more than 8.5 Tris - Citra

te - EDTA buffer (TCE) around 7.0 Tris - EDTAbuffer (TE) around 8.0 Tris - Maleic acid - EDTAbuffer (TME) around 7.5 Lithium Borate - buffer (LB) around 8.6 Supporting me

dium Supporting medium is a matrix in which the protein separation takes place. V ario u s type has b e en u s ed for t h e s e p a rati o n either on slab or capillary fo

rm. Separation is based on charge and mass of protein,depending on the pore size of the medium . 23 Chemical nature Inert Availability easy Electrical conductivity high Adsorpti

vity low Sieving effect desirable Porosity controlled Transparency high Electro - endosmosis(EEO) low Preservation feasible Toxicity low Preparation easy - Starch gel - Cellulos

e acetate - Agarose - Polyacrylamide gel 25 TYPES OF ELECTROPHORESIS 1) Zone Electrophoresis a) Paper Electrophoresis b) Gel Electrophoresis c) Cellulose acetate Electrophores

is d)Isoelectric Focusing e) ImmunoElectrophoresis 2) Moving Boundary Electrophoresis a) Capillary Electrophoresis b)Isotachophoresis 39 PAPER ELECTROPHORESIS  Filter pap

er such as Whatmann no 1 and no 3 mm in strip of 3 or 5 cm wide have been used .  Separation takes place in 12 to 14 hrs. ADVANTAGES:  It is economical.  Easy to use

. DISADVANTAGES:  Certain compounds such as proteins, hydrophilic molecules cannot be resolved properly due to the adsorptive and ionogenic properties of paper which results

in tailing and distortion of component bands .  Electro osmosis. 2  Separation is brought about through molecular sieving technique, based on the molecular size of the sub

stances . Gel material acts as a "molecular sieve” .  Gel is a colloid in a solid form (99% is water).  It is important that the support media is electrically neut

ral.  Different types of gels which can be used are ; Agar and Agarose gel, Starch, Polyacrylamide gels . Gel electrophoresis Agarose Gel (mad e - u p o f r ep e a t uni t of

uni t o f g ala c t o s e and 3 , 6 - ° A line a r pol y sa c ch a ri d e agarobiose - alternating anhydrogalactose). ° Used in conc as 1 % and 3 %. ° The gelling

property are attributed to both inter - and intramolecular hydrogen bonding ° Pore size is controlled by the % of agarose used. ° Large pore size are formed w

ith lower conc and vice versa. ° Purity of the agarose is based on the number of sulphate conc, lower the conc of sulphate higher is the purity of agarose . 30 Resolution is

superior to that of filter paper. Large quantities of proteins can be separated and recovered. I t ad sor b s p r o t e i n s r el a t i v e l y l ess when compar

ed to other medium. 31 a g ar t end t o gi v e di f f e r e n t results and purification is often necessary. Sharp zones are obtained due to less adsorption. APPLIC

ATION: ° To separate DNA, proteins, Hb variants, iso - enzymes etc . DISADVANTAGES:  R e s o l u t i o n i s l e s s co m p a r e d to polyacrylamide gels.  Diffe

rent sources and batches of ADVANTAGES: Easy to prepare and small concentration of agar is required. 4 TYPES OF ELECTROPHORESIS 1) Zone Electrophoresis a) Paper El

ectrophoresis b) Gel Electrophoresis c) Cellulose acetate Electrophoresis d)Isoelectric Focusing e) ImmunoElectrophoresis 2) Moving Boundary Electrophoresis a) Capillary Elect

rophoresis b)Isotachophoresis 39 POLYACRYLAMIDE GEL ELECTROPHORESIS Frequently referred to as PAGE . Cross - linked polyacrylamide gel are formed from the polymerization of t

he monomer in presence of small amount of N,N ” - methylene - bisacrylamide . Bisacrylamide – two acrylamide linked by the methylene group. Th e polym eri z a tion o f

the ac r y l am i d e is an e x ample f o r f r ee radical catalysis. Made in conc. between 3 - 30 % acrylamide. low % has large pore size and vice versa. 43 SDS -

PAGE Sodium do d ec y l sulpha t e - pol y a c ryla m i d e gel electrophoresis. Most wid e ly used m e thod for ana l y s ing protein mixture qualitatively. Useful for m

onitoring protein purification – as separation of protein is based on the size of the particle. Can al so b e us ed for deter m in i ng t h e rel a tive m ole c u l ar

mass of a protein. 45 Mercaptoethanol will break the disulphide bridges. SDSbinds strongly to and denatures the protein. Each protein is fully denatured and open into

rod - shape with series of negatively charged SDSmolecule on polypeptide chain. SDSis an anionic detergent. The sample is first boiled for 5 min in buffer containing • Beta

- M e r ca pto e th a no l • SDS 46 O n a v e r a g e, O n e SD S molecule b i n d f o r e v e r y t w o amino acid residue. Hence original native charge is completely swa

mped by the negative charge of SDS molecule. 47 Components 48 Stacking gel : ° ord e ring/ a rr a ngin g a n d c o n c . t h e m a c r o m ole c ul e b ef or e entering

the field of separation. ( 4 % of acrylamide) ° Purp o s e i s t o c on c en t r a t e p r ot e i n s a m p l e i n sh a r p b a n d before it enters main separating gel.

Running gel : ° the actual zone of separation of the particle/molecules based on their mobility. ( 15 % of acrylamide) Movement of particle 54 [Cl] � [prote

in - SDS] � [Glycinate] 55 - Research tool - Measuring molecular weight - Peptide mapping - Protein identification - Determination of sample purity - Separation of p

roteins and establishing size - Blotting - Smaller fragments of DNA APPLICATION: ADVANTAGES: - Gels are stable o v er wide ra n ge o f p H and temperature. - Gels of diffe

rent pore size can be formed. - Simple and separation speed is good comparatively. - Exhibit reasonable mechanical strength - Low endosmosis effect DISADVANTAGES - Gel pre

paration and casting is time - consuming - carcinogenic - Complete reproducibility of gel preparation not possible STAINING: Fluorescent stains - Ethidium bromide – Nucl

eic acids Silver stain for protein gel (sensitive 50 times dye based ) Dye based – Coomassie blue – protein band Tracking dyes – BPB� xylene cyanol Native (bu

ffer) gel Done by using the polyacrylamide gel ( 7.5 %). SDS is absent. pH of 8.7 Proteins are separated according to the electrophoretic mobility &Sieving effect of the gel

. 60 Isoelectric focussing o f the First described by - H.Svensson in Sweden. Method is ideal for the separation amphoteric substances. Method has high resolution. Able t

o sepa r at e the p r o t ein wh i c h di f f er in i s o el e ct r i c point by as little as little 0.01 of pH unit. 61 Different gradient of the pH along the le

ngth of the separating gel. 62 Establishment of ph gradient: This is achieved by the ampholyte & must have following prop: Must dictate pH course (buffering

capacity at their pI) Should have conductance at their pI. Low molecular weight Soluble in water Low light absorbance at 280nm. Available commercially with pH ba

nd (3 - 11) Eg: Ampholine, Pharmalyte and Bio - lyte. 63 64 M o v e m e n t 65 Application: - Highly sensitive for studying the microheterogeneity of proteins - Useful for

separating the isoenzymes. - Research in enzymology, immunology - Forensic, food and agriculture industry 66 Two - dimensional polyacrylamide gel electrophoresis Principle :

Technique combines with IEFas first dimensional. • Which separate according to the charge. Second dimension by SDS - PAGE • Separate according molecular size. 67 °

T h i s c o m bin a tion gi v es s o ph i s ti c a t ed a n alyti c al m e thod for analysing the protein mixture. ° Size very from 20 * 20 cm to the minigel. ° IEF is

carried on acrylamide gel ( 18 cm* 3 mm), with 8 M urea. ° Af t er sep a r a tio n , pla c ed on 1 0 % S D S - P A GE f o r fu r t her separation . ° Used in field of pro

teomics. ° Can separate 1000 to 3000 proteins from the cell or tissue extract. 68 69 Isotachophoresis ° Used for separation of smaller ionic substances. ° They migrate

adjacent in contact with one another, but not overlapping. ° The sample is not mixed with the buffer prior to run. ° Hence current flow is carried entirel

y by the sample ions. ° Faster moving ions migrate first and the adjacent ones next with no gap between the zone . 71 All ions migrate at the rate of fastest i

on in zones. Then it is measured by UV absorbance. Application - Separation of small anions and cations Amino acids Peptides N uc l eo ti de s N uc l eo s i d e s Protei

ns. Pulsed - Field Electrophoresis Power is applied alternatively to different pair of electrodes Electrophoretic field is cycled at 105 - 180 0 Because of which the molecule ha

veto orient to the new field direction This permit separation of large molecule like DNA. Applied: for typing various strains of DNA. 72 u n der an  Immun o el e ct r o p h

o r e sis r e fe r s to pre c i p itati o n i n a gar electric field.  It is a process of combination of immuno - diffusion and electrophoresis.  A n anti g en mi

x ture is fi r st s e par a ted i n to its com p o n ent parts by electrophoresis and then tested by double immuno - diffusion.  Antigens are placed into wells cut

in a gel (without antibody) and electrophoresed . A trough is then cut in the gel into which antibodies are placed .  The antibodies diffuse laterally to meet diffusing anti

gen, and lattice formation and precipitation occur permitting determination of the nature of the antigens .  The term “immunoelectrophoresis” was first coined by Grabar a

nd Williams in 1953 . Immuno electrophoresis Prin c iple When electric current is applied to a slide layered with gel, antigen mixture placed in wells is separated into individu

al antigen components according to their charge and size . Following electrophoresis, the separated antigens are reacted with specific antisera placed in troughs parallel to the

electrophoretic migration and diffusion is allowed to occur . Antiserum present in the trough moves toward the antigen components resulting in formation of separate precipitin

lines in 18 - 24 hrs, each indicating reaction between individual proteins with its antibody . - Precipitation reactions are based on the interaction of antibodies and antigens.

They are based on two soluble reactants that come together to make one insoluble product, the precipitate (Figure). - These reactions depend on the formati

on of lattices (cross - links) when antigen and antibody exist in optimal proportions. [ it is known as zone of equivalence and appears to us as precipitation]. - Ex

cess of either component reduces lattice formation and subsequent precipitation. Procedure Agarose gel is prepared on a glass slide put in a horizontal position. Using sample te

mplate, wells are borne on the application zone carefully. The sample is diluted 2:3 with protein diluent solution (20μl antigen solution +10 μl diluent). Using a 5 μl pipe

tte, 5 μl of control and sample is applied across each corresponding slit (Control slit and Sample slit). The gel is placed into the electrophoresis chamber with the samples

on the cathode side, and electrophoresis run for 20 mins/ 100 volts. After electrophoresis completes, 20 μl of the corresponding antiserum is added to troughs in moist chambe

r and incubated for 18 - 20 hours at room temperature on a horizontal position . The agarose gel is placed on a horizontal position, and dried with blotter sheets . The gel in s

aline solution is soaked for 10 minutes and the drying and washing repeated twice again . The gel is dried at a temperature less than 70 ° C and may be stained with R e s ults

o Presence of elliptical precipitin arcs represents antigen - antibody interaction. o Absence of formation of precipitate suggests no reaction. o Different antigens (protein

s) can be identified based on the intensity, shape, and position of the precipitation lines . Applications The t e st he l ps i n id e nti f ic a tion a n d a ppro x imate q uan

ti z ation of various antigens in immunology. suspected monoclonal and polyclonal gammopathies. Used to analyze complex protein mixtures containing different antigens. T

he medical diagnostic use is of value where certain proteins are suspected of being absent (e . g . , hypogammaglobulinemia) or overproduced (e . g . , multiple myeloma) . This

method is useful to monitor antigen and antigen - antibody purity and to identify a single antigen in a mixture of antigens. 50 μ m – ID. 300 μ m – ED. Length –

50 - 100 cm. Fused silica capillary tube. Polyimide coating external. Packed with the buffer in use. 82 o Tech n ique fi rst d escri b ed by - Jorgen s en and Lu k a s ( 1

98 0 ’ s ) . A s t h e name suggest, the separation is carried in a narrow bore Capillary Capillary electrophoresis Components : 83 Sample application is done by either of one

method High voltage injection High voltage injection The buffer reservoir is replaced by the sample reservoir the high voltage is applied (+ electrode) buff

er reservoir is placed again and voltage applied for the separation. Pressure i n je c ti o n Pressure injection Anodic end of capillary is removed from buffe

r and placed in air tight sample sol with pressure sample is pushed into capillary kept back in the buffer sample and voltage is applied. 84 ° High voltage

is applied (up to 50 kV) ° The components migrate at different rate along the length . ° Although separated by the electrophoretic migration, all the sample is drawn towards

cathode by electroendosmosis . ° Since this flow is strong, the rate of electro endosmotic flow is greater than the Electrophoretic velocity of the analyte ion, regardless of t

he charge . 85 86 P o s i t i v e l y c h a r g e d m o l e c ul e r e a c h t h e ca th o d e f i r s t (electrophoretic migration + electro osmoticflow). DETECTION: 87 Troub

leshooting : Adsorption of protein to the wall of capillary – leading to smearing of protein – viewed as peak broadening – or complete loss of protein . - Use of neutral c

oating group to the inner surface of the capillary. 88 clinical a ppli ca tion s include Multiple myeloma testing H a e m o g l ob i n o p a t h y screening. H b A 1 c Mo

nitoring chronic alcoholism (GGT). • Online detection. • Improved quantification. • Almost complete automation. • Reduced analysis time. 90 Advantage over conventiona

l Microchip electrophoresis Current advanced method. Development in technique include Integrated microchip design Advanced detection system sepa r a tion c an be New a

pplication P r o t ein and DNA done 91 Instru m e n t a ti o n 92 Similar to the capillary electrophoresis. Separation channel Sample injection ( 50 - 100 pL) Reservoirs

Voltage ( 1 - 4 kV) sample preparation Pre column or post column reactors. Classical Cross - T design. Time period of 50 - 200 sec. Detector : Laser induced fluorescence

Electrochemical detectors Pulsed amperometric detector Sinusoidal voltametry 93 References Tietz - Text book of clinical chemistry. Kaplan - clinical chemistry. YouTube and