Prabin Shah MSc(Biochemistry)

DEFINITIONElectrophoresis is separative technique.

It is the migration of charged partcles in electric field.

The first electrophoretic technique to be widely used was that of Tiselius in 1937.

PRINCIPLEBiomolecules possess ionizable groups

Exist as Cations or Anions depending on pH

Charged particles move to opposite electrode -- cations to cathode

---- anions to anode

Movement based on charge/mass ratio

powerpack

Cathode Buffer Support Buffer Anode

Electrophoresis unit

Requirements Electrophoresis chamber/unitPowerpackBufferStainDestaining solutionFixative solutionSupporting medium

BufferMaintains the pH

pH of the buffer decides the ionic nature of the molecule.

It determines and stabilizes the charges in molecules to be separated.

It also conducts current on the supporting medium.

Buffers in common use are barbitone buffer (pH 8.6, for serum protein

electrophoresis) citrate buffer (pH 6.2 for hemoglobin electrophoresis ) tris-EDTA-borate buffer (pH 8.6,for hemoglobin

electrophoresis).

Power supplyConstant current/constant voltage mode

Low voltage (100 to 500 V ) electrophoresis is routinely used.

High voltage (500 V to 10000 V) electrophoretic technique is employed in the separation of amino acids and small peptides; it requires a cooling system in the electrophoresis unit.

Supporting medium Gel (Agarose, Agar, Polyacrylamide, Starch)Cellulose paper, Cellulose sheet. It should be inert.

Stain :Coomasie brilliant blue / amido black/

Ponceau S – for proteins Ethidium bromide for DNAOil red O – for lipoproteinsDestain : 7 % acetic acidFixative : Methanol/ ethanol

TYPES 1) Moving boundary electrophoresis --- = without supporting medium2) Zone electrophoresis ------ = support medium; horizontal/vertical -- Gel, Paper, Cellulose acetate; --- Isoelectric focussing --- Immunoelectrophoresis

Gel electrophoresisAgar/agarose : Agar is a

heteropolysacchjaride containing agarose and agaropectin. It dissolves in boiling aqueous buffers and forms a gel at 38 C.

Now purified agarose is used. Agarose gel is extensively used for electrophoretic separation of proteins, DNA, isoenzymes, and for immunoelectrophoresis .

Protocol of electrophoresis :

The sample is applied as a narrow line (2-5 microliters) on the support medium, towards the designated cathode end (in case of alkaline pH) , leaving about 1/3rd distance from that end.

The support medium with the sample is kept on the solid support inside the electrophoresis chamber. It is connected to the buffer on both sides with filter paper wicks.

The electrodes are connected to power pack, chamber is closed.

The power is switched on, power supply can be adjusted in constant current ( 7 milliamperes / slide) or constant voltage (100 volts) mode.

Allow the electrophoresis to run for fixed time (alternatively, sample mixed with a dye may be applied and the movement of the dye can be tracked, till it reached the other end of the slide)

After the run, remove the support medium from the chamber, immerse in a fixative(generally methanol or alcohol for proteins) for 15 minutes, the dry it.

For visualization of separated bands, stain with a staining agent. For proteins, coomassie brilliant blue, ponceau S or amido black can be used.

Destain with a washing solution (generally 3% acetic acid), to make the background clear.

Quantitation is possible by densitometry. The slide/cellulose sheet with stained bands is scanned through a densitometer, and a plot of absorbance of each band against the distance moved gives an electrophoretogram.

Based on the height and area of each band/peak, concentration is calculated.

Normal pattern of serum protein electrophoresis :

Albumin; alpha-1, alpha-2, beta ,gamma

globulins

Polyacrylamide Gel ElectrophoresisPAGE : Polyacrylamide gels are prepared

immediately before use from a number of highly toxic, synthetic chemicals. Acrylamide monomer is copolymerized with a cross-linking agent, usually N,N’- methylene bisacrylamide, in the presence of a catalyst ammonium persulfate and initiator TEMED (tetramenthylene diamine).

Gels may prepared containing from 3% to 30% acrylamide, corresponding to pore sizes of 0.5 nm and 0.2 nm respectively. PAGE is most suitable for separation of proteins.

SDS-PAGE is specialized form of PAGE in which a detergent sodium dodecyl sulfate is included. This particularly useful in determination of molecular weights of proteins.

PAGE PATTERN

Cellulose acetate High purity cellulose strips are

commercially available, which have a micropore structure.

Hemoglobin electrophoresis at alkaline pH is best carried on cellulose acetate strip, at a voltage of 450V and the procedure gets completed in half an hour,

Cellulose acetate is also suitable for electrophoresis of glycoproteins and lipoproteins.

Isoelectric focussingAcross the gel layer, voltage and pH

gradients are established. Proteins get focused at their isoelectric pH, as bands.

This technique is very useful in separation of

peptides, proteins, isoenzymes.

ImmunoelectrophoresisThis technique combines together the

specificity of antigen-antibody binding with the electrophoretic technique.

In the quantitative form of immunoelectrophoresis called as Laurell’s rocket electrophoresis, the sample is applied wells cut in agarose gel containing antiserum to the antigen to be assayed .

After the completion of electrophoretic migration, antigen-antibody complexes form as rocket shapes. The area under rocket shape is directly proportional to antigen concentration

Used in diagnosis of immune deificincies, autoimmune disorders, chronic infections -----detect Igs, viral/bacterial antigens.

Rocket electrophoresis

APPLICATIONSIn Clinical diagnosis – serum proteins,

lipoproteins, DNA, Hemoglobin, Enzymes --- infections, immune disorders,

hemoglobinopathies, Lipid abnormalities.In Protein Research --- Isolation,

purification and molecular weight determination

CHROMATOGRAPHYChromatography is collective term referring

to a group of separation processes whereby solutes in a mixture are separated from one another by a differential distribution of solutes between two phases – stationary phase & mobile phase

CHROMATOGRAPHYThis technique received its name from the

work of Tswett in 1906.

He adsorbed a mixture of plant pigments on to finely divided charcoal and then separated the components into a series of coloured bands by washing with solvents.

chromatography ,derived from greek word meaning colour writing.

now it is used for separation of many colourless substances.

A variety of attractive forces between the solutes to be separated and the stationary phase leads to the selective retardation of the stationary phase relative to the mobile phase.

Mobile phase carries the mixture of solutes through the stationary phase.

Stationary phase : solid/liquid supported by glass column/ gel layer/ cellulose paper

Mobile phase : Liquid/ Gas

CLASSIFICATION a) Classification based on principle/

mechanism of separation :i) Partition chromatographyii) ion-exchange chromatography iii)adsorption chromatography Iv) Gel filtration chrromatographyV)Affinity chromatography

Classification based on type of physical apparatus :

Paper, thin layer and column chromatography.

Partition chromatography is carried out on Cellulose sheet(paper), layer of gel (thin-layer) or column .

Ion-exchange, affinity, gel filtration types are carried in columns.

Adsorption chromatography is carried in thin-layer and column modes.

PARTITION CHROMATOGRAPHYThis is based on the separation of solutes

by use of differences in their distribution between two immiscible phases. Separation is based on solubility of solute (the polarity) in two phases.

In liquid-liquid partition chromatography, the both stationary and mobile phases are liquids. In gas-liquid chromatography, liquid is stationary phase and gas is mobile phase.

In liquid-liquid parttion chromatography otherwise simply called as liquid chromatography(LC), solutes are partitioned between stationary and mobile phases, based on distribution coefficient.

Distribution coefficient (kd)/ partition coefficient

= concentration of solute in solvent A concentration of solute in solvent B

In normal phase LC, stationary phase is polar and mobile phase is non-polar. water is the stationary phase; hexane, benzene, chloroform or butanol form the mobile phase.

In reverse phase LC, stationary phase is non-polar (eg. octadecyl silane packing in a column) and mobile phase is polar (solvents like methanol, acetonitrile used in column mode of chromatography).

PAPER chromatographyPartition principleAscending / Descending

Paper chr.--ascending

Paper chromatography.

Chr.chamber

Paper with sample spot

solvent

Solvent for amino acid chr. : butanol:acetic acid:water 12/3/5

TLC(Thin layer chromatography)Silica / Alumina layered over a glass plate -----

thin layerProcedure same as that for paper chr.Better resolution than paper chr.

After the run, Paper/TLC plate

Solvent front

Components

separated as spots/bands

Identification of sample components in paper chr./TLCRf = Relative

front ; =solute front

divided by solvent front

• Run standards parallel with samples

Chr.pattern

HPLC (High-performance liquid chromatography )

conducted in a column; use of high pressure and smaller particle size when compared to routine LC techniques, gives greater resolution.

HPLC can be run in normal as well as reverse phase modes and any principle of chromatography can be used in this (partition/affinity/gel filtration/ion exchange).

It is the most versatile form of chromatography used for analysis of wide variety of compounds.

GAS CHROMATOGRAPHY(GC)Gas is the mobile phase and liquid/solid is the

stationary phase. The sample is injected into a stream of inert

gas usually at an elevated temperature.

The vapourized sample is carried into a column packed with the stationary phase.

ION-EXCHANGE CHROMATOGRAPHY

Separates molecules based on charge.

Electrostatic attraction

Mobile phaseGenerally liquid

Stationary phaseElectrostatically

charged ions bound to insoluble, chemically inert martrix.

Elution of Molecules

Ion-exchange resinsH2C

C

O

O-

carboxymethyl (CM)

cellulose

H2C

CH2

N

CH2

CH3

H2C

CH3

H

diethylaminoethyl (DEAE)

cellulose

ADSORPTION CHROMATOGRAPHYAdsorption is a process whereby one

substance adheres to another because of attractive forces between surface atoms .

In adsorption chromatography, adsorbents are used. Examples of adsorbents are charcoal, silica gel and alumina.

This form of chromatography can be carried out both in thin-layer and column modes.

Affinity chr.This is based on

strong covalent binding between a macromolecule and a ligand.

Eg. of molecule and ligand----

Enzyme-substrate Hormone-receptorAntigen-antibody

Gel filtration/ exclusion/molecular sieve chr.Separates molecules

based on size.

Large molecules exit first.

Mobile phaseLiquid

Stationary phaseInsoluble, porous

carbohydrate beads

Applications of chr.• In clinical diagnosis : detection and

estimation of of amino acids, metabolites, sugars, mucopolysaccharides in urine and blood –useful for screening and diagnosis of inborn metabolic disorders

: Aminoacidurias, hemoglobinopathies, mucopolysaccharidoses, etc.

In clinical diagnosisPaper chr.,TLC –qualitativeHPLC, GC –For quantitationIn clinical diagnosis Assay of Hormones, drugs, vitamins,

metabolites ---HPLC and GC

Applications of chr.Chromatography in protein research –1) Purification –adsorption, affinity, gel

filtration chr.2) Sequencing – ion-exchange chr.3) Mol.wt. determination – gel filtration chr.

PLASMA PROTEINSALBUMINGLOBULINSFIBRINOGEN

SEPARATION METHODSSalt fractionation – Sodium sulfite, sodium

sulfate, ammonium sulfateAlcohol fractionationElectrophoresis --- Main five fractions

Plasma proteins in each fractionAlpha-1 globulins Alpha-1 antitrypsin

Alpha-1 acid glycoprotein

Alpha lipoprotein

Alpha-2 globulins CeruloplasminAlpha-2

macroglobulinHaptoglobin

Beta-globulins Beta-lipoproteinTransferrinHemopexinSome Igs

FUNCTIONS1)Maintenance of colloidal osmotic

pressure(COP)—80% by albumin*In arterioles *In

Venules

COP 25mm Hg

BP 35mm Hg

BP 15mmHg

functions2) Transport3) Nutritional4) Buffering5) Defense –clotting,

immunoglobulins6) Viscosity

Albumin– free fatty acids, steroid hormones, bilirubin, copper

Transferrin –ironRetinol-binding

protein : vitamin ALipoproteins : lipids

ALBUMINSynthesised in liver; 12gm/day60% of total protein in plasma585 amino acids; elipsoidal shapeMol.wt. 69,000COP maintenance, transport protein,

buffering

Transport proteinsTransferrinRBPTBPA TBGTRANSCORTINHAPTOGLOBINHEMOPEXIN

Acute phase reactantsIncreased during

acute inflammatory states or secondary to certain types of tissue damage

C-reactive proteinAlpha-1 antitrypsinHaptoglobinAlpha-1 acid

glycoproteinfibrinogen

Plasma protein abnormalitiesHypoalbuminemiaAnalbuminemiaHyperalbuminemia

Liver cirrhosis,malnutrition

Nephritis, burns, protein losing enteropathies

Fever, starvation, Diabetes mellitus

Plasma protein abnormalitiesHYPER

GAMMAGLOBULINEMIAS

Hypogamaglobulinemia

Agammaglobulinemia

Chronic infectionsMultiple myeloma

Electrophoretic patterns1) Myeloma : M band2) Nephrotic syndrome : increased alpha-2,

decreased albumin3) Liver disease : decreased albumin, beta-

gamma bridging4) Chronic infections : broad-based increase

in gamma globulins

IMMUNOGLOBULINS4 polypeptide chains – 2 light and 2 heavy

Light chains – 2 kappa (k) or

2 lamda (λ)chains ;

Heavy chains – 2 chains of any of the five types ---

α , γ, δ, ε, or µ type

Immunoglobulins -types5 types : classification based on type of heavy

chain present –1) IgA : α heavy chain 2) IgG : γ heavy chain3) IgD: δ heavy chain4) IgM: µ heavy chain5)IgE: ε heavy chain

Igs – properties and functionsIgA :1) Ig of secretions –dimeric 2) also present in plasma3) Secondary immune response4)Agglutination and compliment fixation

IgG :Smallest IgHighest plasma concentration(75% of Ig in

plasmaSecondary immune responsePlacental transferAgglutinationCompliment fixation

IgM :Largest Ig; pentamericPrimary immune responseNatural anibodyAgglutination ++++Compliment fixation

IgE:1)Fix On mast cells & basophils2)Mediate anaphylaxis 3)Igs of allergic reactions and response to

worm infectionsIgD : No well known fn., found on surface of

B lymphocytes

Multiple myelomaProliferation of plasma cellsParaproteinemiaAbnormal Igs in plasmaOnly heavy chain /only light chainImmunodeficiencyIncreased total protein, M bandBence Jones Proteinuria