THEORY AND APPLICATION OFISO-TACHOPHORESIS AND ISOELECTRIC FOCUSSING.

PRESENTED BY,VINEETHA K

1ST M.PHARM DEPT. OF PHARMACEUTICS

NGSMIPS.

CONTENTS ISOELECTIC FOCUSSING PRINCIPLE THEORETICAL ASPECTS PREPARATION OF IEF GEL DETERMINATION OF ISOELECTRIC POINT OF

PROTEIN APPLICATION ISOTACHOPHORESIS PRINCIPLE APPLICATION.

ISOELECTRIC FOCUSSING Electrophoretic method that separates proteins

according to the iso-electric points.

Is ideal for separation of amphoteric substances.

Separation is achieved by applying a potential difference across a gel that contain a pH gradient.

Isoelectric focusing requires solid support such as agarose gel and polyacrylamide.

Isoelectric focusing gels contain synthetic buffers called ampholytes that smooth the pH gradients.

Ampholytes are complex mixtures of synthetic polyamino-polycarboxylic acids

Commercially available ampholytes are- BIO-LYTE PHARMALYTE

It gives good separation with a high resolution compared to any other method

Resolution depends on

I. The pH gradient, II. The thickness of the gel III. Time of electrophoresis, IV. The applied voltage, V. Diffusion of the protein into the gel

pI:Isoelectric focusing uses the theory of protein pI.

pI is the pH at which a given protein has a neutral overall charge.

The pI is dependant on which type of residues are present and how many bases make proteins positive and acids negative.

pI is very specific for each protein.

PRINCIPLEIsoionic point is the point at which the net charge of protein

molecule is zero.

In isoelectric focusing a stable pH gradient established.

pH increases gradually from anode to cathode.

A protein introduced at a point where the pH is lower than the isoionic point will posses a net positive charge & will migrate in the direction of cathode.

Due to the presence of protein gradient, the protein will migrate to an environment of higher pH values .

Iso electric point of protein varies with the environment.

Isoelectric point is taken to be the pH where all the an ions on the protein are exactly balanced by equal number of cations;the net charge is zero & the protein does not migrate in the electric field.

The value of pH provides a rough idea about the amino acid composition of a given protein.

•Finally protein will encounter a pH where its net charge is zero & will stop migrating.

•This is the isoelectric point of protein.

THEORETICAL ASPECTS.The pH gradient forces a protein to remain in its isoelectric

point position, thus concentrating it ; this concentrating effect is called "focusing".

Increasing the applied voltage or reducing the sample load result in improved separation of bands. The applied voltage is limited by the heat generated, which must be dissipated.

The use of thin gels and an efficient cooling plate controlled by a thermostatic circulator prevents the burning of the gel whilst allowing sharp focusing.

The separation is estimated by determining the minimum pI difference (ΔpI), which is necessary to separate 2 neighboring bands:

What Happens

Proteins stop exactly at pH=pI and the stained proteins are very visible

1,4

PREPARATION OF IEF GEL Carrier ampholytes (suitable pH) and riboflavin mixed with

acrylamide solution.

Mixture is poured over a glass plate which contain spacer

Second glass plate is placed on first

Gel is polymerized

This takes 2-3 hr

After the gel has set glass plates are prised apart

Electrode wicks are laid along the long length of each side of the gel

Potential difference is applied

Ampholytes form a pH gradient between anode and cathode

The power is then turned off

Samples applied by laying on gel filter paper soaked in the sample

Voltage is again applied for 30 min

Proteins having positive charge will migrates towards the cathode. negatively charged protein

will migrates towards anode

Become stationary when they reaches isoelectric point

The gel is washed with trichloroacetic acid

This precipitaes the proteins and allows smaller ampholytes to be washed out

Gel is stained with Coomasie Brilliant Blue

Destained

A TYPICAL ISOELECTRIC FOCUSING GEL

DETERMINATION OF ISOELECTRIC POINT OF PROTEIN.

To determine isoelectric point of protein (polypeptide),one has to:

i. Measure the mobility of protein at several different pH values.

ii. Plot the mobility values so obtained against the pH values.

iii. Take reading of pH of that point where the point intersects zero mobility line.

The pH at this intersection is then taken to be the isoelectric point of the concerned protein.

Diagrammatic representation of the principle of pH determination. The pH at zero mobility is the pI.

APPLICATION Useful for separating, purifying &

characterizing proteins. Identification of iso enzymes. Separation & identification of serum

proteins.

Used by food & agricultural industries, forensic & human genetic laboratories.

Used for research in enzymology, immunology & membrane biochemistry.

ISO-TACHOPHORESISThis method is based on the principle of moving boundary

electrophoresis.

Separation is achieved either in a horizontal or vertical way based on the equipment employed.

The name iso-tachophoresis is derived from Greek , refers to the fact that the ions being separated all travel (phoresis) at the same( iso)speed( tacho).

Separation of ionic components of sample is achieved through stacking them into discrete zones in order of their mobilities, producing very high resolution.

Migration of small ionsDiscontinuous electrolyte system

leading electrolyte (L- ions) & trailing electrolyte (T- ions)

Apply sample solution at interphase of L & TApply electric field -> each type of ion arrange

between L and T ions -> discrete zonesSeparates small anions, cations, organic & amino

acids, peptides, nucleotides, nucleosides, proteins

Separation of mixture of an ions involves:

A leading anion (chloride) with higher mobility than the sample ion.

A trailing an ion (glutamate) with lower mobility than the sample ion.

A common cat ion for all the anions.

When the current is switched on, the leading ions will move towards appropriate electrode.

The sample ions will follow in order of their mobilities .

The trailing will follow the sample ions.

Once equilibrium is achieved ions will all move at the same speed in discrete bands in order of their mobilities.

Where the mobilities of sample ions are similar, the resolution can be enhanced by including with the sample, synthetic ampholytes called spacer ions.

These have mobilities intermediate to that of sample ions and help to separate them by taking up positions between the sample ions.

The spacer ions are similar to ampholytes used in isoelectric focusing.

APPLICATIONUsed for separating charged substances ranging from inorganic to

organic acids to proteins and nucleic acids.

Used extensively in research laboratories.

Find applications in industries in pollution control(detecting detergents and inorganic ions in effluent water)

In quality control in the food, brewing and pharmaceutical industries.

Samples as small as few micrograms can be separated quantitatively,& large quantities of sample can be separated preparatively .

REFERENCES:

Pictures:2. http://www.science-tube.com/ 3. http://www.zeitnews.org/4.http://www.biochem.arizona.edu/classes/

bioc462/462a/NOTES/Protein_Properties/protein_purification.htm

3 .Introduction to chemical analysis, by “H.K.Kaur4.S.M. Supriya .Instrumental methods of

analysis.1st edn.India:Popular prakashan;2010.343-347

1. Voet, D. Voet, J. G. Pratt. C. W. Fundamentals of Biochemistry: Life at the Molecular Level. 3rd edition. John Wiley and Sons. (2008)

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