Protein fractionation is a process used to isolate individual protein components from a complex protein mixture. It involves separating the mixture into fractions based on differences in physical properties like charge, size, or affinity for specific ligands. Various techniques can be used for fractionation including precipitation, centrifugation, chromatography, electrophoresis, and ultrafiltration. The document provides details on these techniques and how they utilize differences in properties like solubility, isoelectric point, or molecular weight to separate protein mixtures into purer fractions.

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2. Protein Fractionation
Presented By:
Jaspreet kaur
Dep. Of Food Science & Nutrition

3. Content:
3
Introduction & Definition of Protein
Fractionation
Fractionation protocol for Protein
Strategy of Fractionation
Protein Fractionation Techniques:
Conclusion
References

4. Introduction
Fractionation is a separation process in which a
certain quantity of a mixture (solid, liquid,
suspension or isotope) is divided up in a number
of smaller quantities (fractions) in which the
composition varies according to a gradient.
Fractions are collected based on differences in a
specific property of the individual components. A
common trait in fractionations is the need to find
an optimum between the amount of fractions
collected and the desired purity in each fraction.
Fractionation makes it possible to isolate more
than two components in a mixture in a single run.
This property sets it apart from other separation
techniques.4

5. Introduction
Fractionation is a separation process in which a
certain quantity of a mixture (solid, liquid,
suspension or isotope) is divided up in a number
of smaller quantities (fractions) in which the
composition varies according to a gradient.
Fractions are collected based on differences in a
specific property of the individual components. A
common trait in fractionations is the need to find
an optimum between the amount of fractions
collected and the desired purity in each fraction.
Fractionation makes it possible to isolate more
than two components in a mixture in a single run.
This property sets it apart from other separation
techniques.5

6. 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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7. 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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8. 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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9. Strategy of Fractionation
Fractionation 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

10. 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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11. Protein fractionation
techniques:
Homogenisation
Centrifugation
Fractionation by precipitation
Ultra filtration
Chromatographic and
Electrophoresis
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12. Homogenisation
Hydrophilic 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
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13. 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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14. 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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15. 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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16. 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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17. 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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18. 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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19. 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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20. 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 to
size through the use of semi permeable membranes
containing pores of less than macromolecular
dimensions.
Pores in the membrane allow solvents, salts and small
metabolites to diffuse across but block larger molecules.
Cellophane (cellulose acetate) most commonly used
dialysis material.
Usually used to change the solvent in which the protein is
dissolved in.
Can also be used to concentrate a protein solution by20

21. Figure : Use of dialysis to separate
small and large molecules.
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22. 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.
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23. 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

24. 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

25. •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.
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Types of chromatography

26. 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

27. 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.

28. Cont….
PRINCIPLE
Reversible electrostatic
attraction of a charged
molecule to a solid matrix
possessing opposite charge.
Elution is done by increasing
salt concentration or changing
pH.
FACTS
Proteins possess both (+) and
(-) charges
At pH=7:
Aspartic and glutamic acid
have negatively charged side
groups28

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30. 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).

31. 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

32. 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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33. Affinity chromatography
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34. 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.

35. 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

36. 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 nonpolar
mobile 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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38. 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

39. 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.

40. Thin Layer Chromatography

41. 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.

42. 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

43. 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 polymerase
Steps 1 2 3 4 5 6
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44. Conclusion
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45. ANY
QUESTIONS?
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46. 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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