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Protein Fractionation

Presented By:Jaspreet kaur

Dep. Of Food Science & Nutrition

Content:

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Introduction & Definition of Protein

Fractionation

Fractionation protocol for Protein

Strategy of Fractionation

Protein Fractionation Techniques:

Conclusion

References

IntroductionFractionation is a separation process in which acertain quantity of a mixture (solid, liquid,suspension or isotope) is divided up in a numberof smaller quantities (fractions) in which thecomposition varies according to a gradient.Fractions are collected based on differences in aspecific property of the individual components. Acommon trait in fractionations is the need to findan optimum between the amount of fractionscollected and the desired purity in each fraction.Fractionation makes it possible to isolate morethan two components in a mixture in a single run.This property sets it apart from other separationtechniques.

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IntroductionFractionation is a separation process in which acertain quantity of a mixture (solid, liquid,suspension or isotope) is divided up in a numberof smaller quantities (fractions) in which thecomposition varies according to a gradient.Fractions are collected based on differences in aspecific property of the individual components. Acommon trait in fractionations is the need to findan optimum between the amount of fractionscollected and the desired purity in each fraction.Fractionation makes it possible to isolate morethan two components in a mixture in a single run.This property sets it apart from other separationtechniques.

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Protein Fractionation

Definition

Protein Fractionation is a process or series of

processes intended to isolate a single or multiple

type of protein from a complex mixture of

proteins.

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Fractionation of food

Fractionation is also used for culinary purposes, as

coconut oil and palm oil are fractionated to produce

oils of different viscosities, that may be used for

different purposes. Mango oil is an oil fraction

obtained during the processing of mango butter.

Milk can also be fractionated to recover the milk

protein concentrate or the milk basic proteins fraction.

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Fractionation protocol for

Protein

Prerequisite information about the protein.

Protein are diverse in composition structure

behaviour, you should know about their origin. As your

fractionation strategy depends on it.

1. Where is this enzyme or protein present in the

cell?(intracellular, extracellular, membranous).

2. How you can purify this protein in as few steps as

possible without the loss of activity.(assayable

enzyme activity).

3. Keeping in consideration of temperature and time.

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Strategy of FractionationFractionation procedures or steps to isolate protein based on physical

characteristics.

Characteristic Procedure

Charge 1. Ion exchange

2. Electrophoresis

3. Isoelectric focusing

Polarity 1. Adsorption chromatography

2. Paper chromatography

3. Reverse phase

chromatography

4. Hydrophobic interaction9

cont…Characteristic Procedure

Size 1. Dialysis

2. Gel electrophoresis

3. Gel filtration

4. Ultracentrifugation

Specificity 1. Affinity chromatography

2. Immunopurification

Solubility 1. Salt precipitation

2. Detergent solubilization

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Protein fractionation

techniques:

Homogenisation

Centrifugation

Fractionation by precipitation

Ultra filtration

Chromatographic and

Electrophoresis

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HomogenisationHydrophilic peptides/protein are generally extracted with

homogenization in water or in solutions of organic acids

whereas organic solvents are used to obtain highly

hydrophobic peptides/protein.

Homogenization in a mixture of organic solvents

(chloroform/ methanol) can be used for peptide extraction

as well as for the removal of sample interferences after

producing a biphasic system.

By using this method, Kostyra et al., (2003 ) studied the

opioid activity of cheese and fermented milk samples and

some other samples, homogenization in water has been

widely applied on cheese, fish, meat, and cereals samples

Typically, the ratio of water to cheese used was 2:1 in the

homogenization process, followed by an incubation step of

an hour at 60°C.

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Conti.....Homogenization

Homogenization in a blender and in a Dounce are

common and simple procedures form disruption soft

tissues such as liver, heart, brain, and muscle or other

sample.

These methods are rapid (5 to 10 min) and gentle to

proteins.

These homogenization procedures usually produce

heat, and thus the blender and the associated

container should be prechilled at 4℃.

The homogenization should be performed in a cold

room or on ice.

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Cont….

To get a homogeneous solution after getting rid of cell

debris, tissues and insoluble stuff either by filtering

through muslin cloth or filter paper. This is called

Crude extract containing the protein or enzyme of

your interest plus a mixture of other proteins.

Find out the amount of total protein(By Protein assay)

in this Crude extract.

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Protein Assay

Lowry method as being the most accurate and sensitive

method down to 0.01mg/mL and widely used .

Based on the biuret reaction in which the peptide

bonds of the proteins react with copper under

alkaline conditions to produce CU+, which reacts with

the Follins reagent resulting in strong blue colour

which depends partly on aromatic a.acid such as

tyrosine and tryptophane.

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Centrifugation

The use of centrifugation is one of the simplest

methods used for isolation and fractionation of

proteins. Centrifugation can be used for different

purposes.

It can be a first step to separate different cell

substructures where our proteins of interest are

locally concentrated, for instance, mitochondria,

membrane, or nucleus.

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Conti.....

This process involves multiple centrifugation steps

and, as a result, the cellular homogenate is

separated into different layers based on the

molecular weight, size, and shape of each

component. Afterwards, solubilization steps and

enrichment and fractionation steps should be

carried out to isolate the protein fraction from the

selected layer prior to MS analysis.

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Centrifugation

Separate proteins by size or density

Differential centrifugation - separates large from small particles

Isopycnic (sucrose-density) centrifugation - separates particles of different densities

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Precipitation

It is recognized that among the different precipitants

the most widespread is ammonium sulphate.

The addition of high amounts of this salt or other such

as sodium chloride into a protein solution provokes an

increase of protein interactions followed by protein

aggregation and finally precipitation. This is known as

a salting-out process and, as the salt concentration

needed for protein precipitation varies from one

protein to another, it allows selective protein

separation.

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Salt Out

Dialysis is commonly used for removing the salt from the

proteins. As ammonium sulphate presence in the protein

can interfere in many ways.

Dialysis-a process that separates molecules according tosize through the use of semi permeable membranescontaining pores of less than macromoleculardimensions.

Pores in the membrane allow solvents, salts and smallmetabolites to diffuse across but block larger molecules.

Cellophane (cellulose acetate) most commonly useddialysis material.

Usually used to change the solvent in which the protein isdissolved in.

Can also be used to concentrate a protein solution byplacement in a polymeric dessicant (PEG) which cannot

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Figure : Use of dialysis to separate

small and large molecules.

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Ultra Filtration

Ultra filtration is mainly useful for fractionating

peptides as well as the removal of proteins and

other macromolecules based on their molecular

size. Dedicated membranes are mostly made of

polysulfone or cellulose derivatives.

UF is used extensively in the dairy industry;

particularly in the processing of cheese whey to

obtain whey protein concentrate (WPC) and

lactose-rich permeate.

In a single stage, a UF process is able to

concentrate the whey 10-30 times the feed.22

Chromatography

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Analytical methods used to separate molecules.

Involves a mobile and a stationary phase.

• Mobile phase is what the material to be separated

is dissolved in.

• Stationary phase is a porous solid matrix which

the mobile phase surrounds.

• Separation occurs because of the differing

chemistries each molecule has with both the

mobile and stationary phase.

• Chemistries are different depending on the

specific method.

Chromatography terms

The analyte is the substance to be separated

during chromatography.

The eluate is the mobile phase leaving the

column.

The eluent is the solvent that carries the analyte.

The sample is the matter analyzed in

chromatography. It may consist of a single

component or it may be a mixture of

components.

•Gas - Solid: Mobile phase is gaseous, stationary

phase is a solid matrix.

•Liquid - Solid: Mobile phase is liquid, stationary

phase is a solid matrix.

If separation is based on ionic interaction the

method is called Ion Exchange chromatography.

If separation is based on solubility differences

between the phases the method is called

adsorption chromatography.

If the separation is base on size of molecule the

method is called gel filtration or size exclusion.

If the separation is base on ligand affinity the

method is called Affinity chromatography.25

Types of chromatography

Some other Important

Chromatography

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Paper Chromatography: In this system a filter paper

sheet is used as support for stationary phase.

Thin Layer chromatography: In this technique a

glass plate, plastic sheet or a piece of metal foil

serves as a support for the stationary phase which is

applied in the form of a thin layer on these material.

Colum chromatography: in this technique stationary

phase is packed into a tubular glass or metal column

Ion-exchange chromatography

Ion-exchange chromatography (or ion

chromatography) is a process that allows the

separation of ions and polar molecules based on their

charge. It can be used for almost any kind of charged

molecule including large proteins, small nucleotides

and amino acids. It is often used in protein

purification, water analysis, and quality control.

Cont….

PRINCIPLE

Reversible electrostaticattraction of a chargedmolecule to a solid matrixpossessing opposite charge.

Elution is done by increasingsalt concentration or changingpH.

FACTS

Proteins possess both (+) and

(-) charges

At pH=7:

Aspartic and glutamic acid

have negatively charged side

groups

Lysine, arginine, histidine

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Gel Filtration Chromatography

Gel filtration (chromatography), is also known as molecular sieve chromatography.

Gel filtration chromatography separates molecules according to their size and shape.

The stationary phase consists of beads containing pores that span a relatively narrow size range.

Smaller molecules spend more time inside the beads than larger molecules and therefore elute later (after a larger volume of mobile phase has passed through the column).

Theory

Column matrix

Porous beads

Large molecules are “excluded” from the pores

and travel through the column fastest

Small molecules are “included” – can diffuse

into the pores and elute later

Affinity Chromatography

Described as the most powerful highly selective

method

It relies on the ability of most proteins to bind

specifically and reversibly to their ligands.

Generally used in late purification steps.

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Affinity chromatography

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Column chromatography

Column chromatography (CC) is an extremely

valuable technique for purification of synthetic or

natural products.

Compounds are separated by CC through the

same mechanism as TLC; through differential

intermolecular forces between the components

of the mixture with the mobile phase, and

between the components with the stationary

phase.

A variety of adsorbents can be used as the

stationary phase; silica gel (which is very polar)

is most commonly used in organic chemistry.

Polar components (b) adsorb more strongly to the polar silica and elute after

the less polar components (a), which move more quickly with the non-polar

(relative to silica) solvent.

COLUMN CHROMATOGRAPHY

Paper Chromatography

Paper chromatography - separation of small polar molecules. Mostly used to separate amino acids, oligopeptides. Historically the first chromatography but not really used today. However, principles of its use are useful to know.Rates of migration of the substances are determined by relative solubilities in the polar stationary phase (paper) and the nonpolarmobile phase

• A given solute is distributed between the mobile and stationary phases according to its partition coefficient

Kp =concentration in stationary phase

concentration in mobile phase

Molecules are separated according to their polarities, with nonpolar

molecules moving faster than polar molecules

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Conti….After the solvent has migrated an appropriate distance, the chromatogram is removed from the solvent and dried. If not colored, the separated materials can be detected by radioactivity, fluorescence, etc.

The migration rate of the substance is expressed by the following ratio:

Distance traveled by substance

Rf = Distance traveled by solvent front

Each substance has a characteristic Rf value for a given solvent and paper type.38

Thin Layer Chromatography

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Principle: In thin layer chromatography separation of a

mixture is achieved over a thin layer of aluminum

oxide or silica gel to which they are adsorbed by

different physical forces.

Thin layer chromatography (TLC) is a chromatography

technique used to separate mixtures. Thin layer

chromatography is performed on a sheet of glass, plastic,

or aluminium foil, which is coated with a thin layer of

adsorbent material, usually silica gel, aluminium oxide, or

cellulose (blotter paper). This layer of adsorbent is known

as the stationary phase.

Thin Layer Chromatography

Electrophoresis Methods

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Electrophoresis separates mixtures of proteins based

on charge, charge/mass ratio or size.

Principle

1. It is the process of moving charged biomolecules in

solution by applying an electrical field across the

mixture.

2. Biomolecules moved with a speed dependent on

their charge, shape, and size and separation

occures on the basis of molecular size.

When charged molecules are placed in

an electric field, they migrate

toward either the positive (anode)

or negative (cathode) pole

according to their charge.

1. Factors influenced electrophoresis

mobility:

2. net charge of the molecule

3. size and shape

4. concentration of the molecule in

solution

Electrophoresis

Separation of proteins, nucleic acids, etc. by size, shape, charge

Proteins migrate based on their charge-to-mass ratio

Proteins visualized (radioactivity or staining)

Use gels made of crosslinked polymer (polyacrylamide) or solidified agarosePurification of RNA polymeraseSteps 1 2 3 4 5 6

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Conclusion

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ANYQUESTIONS?

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References

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http://www.google.com/search?q=affinity+chromatogr

aphy+%3B793%3B528

http://www.google.com/webhp?nord=1#nord=1&q=el

ectrophoresis

http://www.sigmaaldrich.com/catalog/product/sigma/d

9938?lang=en&region=IN

Sawhney S.K. and Randhir Singh (2011).Intoductory

Practical Biochemistry.9th Reprint pp 195-216.

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