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NAME MANI UPADHYAY
ROLL NO 2102977
CLASS Bsc(ABS)IV SEM
SUB CODE BOM 305
SUBMITTED TO
DR.ANURADHA PATEL
TECHNIQUE OF
PROTEIN
PURIFICATION
Proteins are very large molecules composed of basic
units called amino acids. Proteins contain carbon,
hydrogen, oxygen, nitrogen, and sulphur.
Protein molecules are large, complex molecules formed
by one or more twisted and folded strands of amino
acids
WHAT IS PROTEIN
WHYWE NEEDTOPURIFY
PROTEIN?
a.TO IDENTIFY STRUCTURE AND FUNCTION OF PROTEIN OF
INTEREST
b.TO STUDY PROTEIN REGULATION AND PROTEIN-
INTERACTIONS
c.TO PRODUCE ANTIBODIES
d.PROTEINS ARE USED IN ASSAYS
e.PERFORM STRUCTURAL ANLYSIS BY X-RAY
CRYSTALLOGRAPHY
Proteinprecipitation
The main purpose of protein precipitation is to
separate the protein from the solution either to
eliminate interferences or to purify them.
Hydration shell plays a very important role in
protein folding and function and is the target of
all protein precipitation methods .It is the
surrounding water that is formed around
proteins, also referred to as “protein hydration”
1. Acid precipitation - This method relies on
the changes of a solution pH. Every protein has a
defined isoelectronic point or pI value, little changes
in a medium pH have an impact on the protein
structure. Adding acids to the solution, lowers the
pH and leads to positively charging the protein In
an aqueous solution the hydration sphere
surrounding a protein is disrupted; occurred
imbalance in structure leads to precipitation. Most
popular acid used in this method is TCA
(Trichloroacetic Acid),
2. Salting precipitation - Adding the salt ions
into the solution cause the restriction of the
available water molecules for the proteins,
which leads to destruction of the hydrogen
bonds. The interaction between proteins is
stronger than between the protein and the
available water molecules which causes
the protein aggregation and precipitation.
Examples are zinc sulphate and ammonium
sulphate. This method is widely used for initial
fractioning of different proteins, based on their
solubility.
3. Alcohol precipitation - Organic
solvents can be also used. Adding alcohol to
the solution reduces the hydration of the
protein, by removing water and surrounding
proteins which leads to aggregation and
precipitation.
To preserve their biological functions, it is
CHROMATOGRAPHY
In this process, we apply the mixture to be separated on a stationary phase (solid or liquid) and a pure
solvent such as water or any gas is allowed to move slowly over the stationary phase, carrying the
components separately as per their solubility in the pure solvent.
TYPES OF CHROMATOGRAPHY
1. Thin Layer Chromatography
In the process of thin-layer chromatography (TLC), the mixture of
substances is separated into its components with the help of a glass
plate coated with a very thin layer of adsorbent, such as silica gel
and alumina, as shown in the figure below.
The plate used for this process is known as chrome plate. The
solution of the mixture to be separated is applied as a small spot at a
distance of 2 cm above one end of the plate. The plate is then
placed in a closed jar containing a fluid termed as an eluant, which
then rises up the plate carrying different components of the mixture
to different heights.
2. Column Chromatography
Column chromatography is the technique used to
separate the components of a mixture using a
column of suitable adsorbent packed in a glass
tube, as shown in the figure below. The mixture is
placed on the top of the column, and an appropriate
eluant is made to flow down the column slowly.
Depending upon the degree of adsorption of the
components on the wall adsorbent column, the
separation of the components takes place. The
component with the highest absorptivity is retained
at the top, while the other flow down to different
heights accordingly.
DIALYSIS
‘Dialysis’ is a process of separating PROTEIN molecules or other
molecules in particular solutions with the help of a membrane that is
semipermeable known as a dialysis tube.
It is also used in the studies of drugs and electrophoresis. In dialysis a
semipermeable membrane is used to separate small
molecules and protein based upon their size. A dialysis bag
made of a semipermeable membrane (cellulose) and has
small pores. The bag is filled with a concentrated solution
containing proteins. Molecules that are small enough to pass
through the pores of the membrane diffuse out of the bag
into the buffer solution, or dialysate. Dialysis is sometimes
used to change buffers. The molecules go from an area of
high concentration to low concentration. When the level of
concentration is equal between the bag and the buffer, there
is no more net movement of molecules. The bag is taken out
and inserted into another buffer, causing the concentration to
be higher in the bag relative to the buffer. This causes more
diffusion of molecules. This process is repeated several
times to ensure that all or most of the unwanted small
molecules are removed (usually done overnight). In general,
dialysis is not a means of separating proteins, but is a
method used to remove small molecules such as salts. At
equilibrium, larger molecules that are unable to pass through
the membrane remain inside the dialysis bag while much of
the small molecules have diffused out.
Lyophilization
If the solution doesn't contain any other
soluble component than the protein in
question the protein can
be lyophilized (dried). This is commonly
done after an HPLC run. This simply
removes all volatile components, leaving
the proteins behind.
Electrophoresis
THIS TECHNIQUE SEPARTES PROTEINS
BASED ON SIZE UNDER AN ELECTRIC
FIELD AND CAN BE USED FOR FURTHER
PURIFICATION AFTER
CHROMATOGRAPHY STEPS UNLESS
RESOLUTION OF CLOSELY REALTED
SPECIES IS REQUIRED
1. PAPER ELECTROPHORISIS
PAPER ELECTROPHORISIS IS ONE OF THE TYPE OF
ZONE ELECTROPHORESIS
PROCEDURE
.
• Using an electric field, molecules (such as DNA) can be
made to move through a gel made of agar or
polyacrylamide.
• The electric field consists of a negative charge at one end
which pushes the molecules through the gel, and a
positive charge at the other end that pulls the molecules
through the gel.
• The molecules being sorted are dispensed into a well in
the gel material.
• The gel is placed in an electrophoresis chamber, which is
then connected to a power source .
• When the electric current is applied, the larger molecules
move more slowly through the gel while the smaller
molecules move faster.
• The different sized molecules form distinct bands on the
gel.
WHERE DO WE GET
PROTEINS
1. WHOLE ORGANISM
2. ORGANS OR TISSUE
3. EMBRYOS
4. PROTEINS FROM EXPRESSION SYSTEM
(GENES CLONED INTO A VECTOR , INSERTED INTO A
LIVING ORGANISM WHICH MAKES THE PROTEIN)
THE BASIC AIM IN PROTEIN PURIFICATION IS
TO ISOLATE ONE PARTICULAR PROTEIN OF
INTEREST FROM OTHER CONTAMINATING
PROTEINS TO STUDY ITS STRUCTURE AND
FUNCTION , INCREASING ITS STABILITY AND
LARGE SCALE PRODUCTION .
APPLICATIONOF PURIFIED
PROTEINS
1.HORMONES – INSULIN ,
ERYTHROPOIETIN , CLOTTING
FACTORS
2.DIAGNOSTICS – LFT , KFT , TFT
3.CELLULAR MARKERS – CANCER
4.IN RECOMBINANT DNA TECHNOLOGY
TO LOCATE GENE OF INTEREST
5.VACCINES
6.SUPPLEMENTS
ISOLATIONOFPROTEINS
1. METHOD OF ISOLATION OF PROTEINS DIFFRS FROM ONE
PROTEIN TO OTHER .
2. PROTEINS CAN BE ISOLATED ON THE BASIS OF FOLLOWING
PROPERTIES :-
CHACTERISTICS PROCEDURE
SOLUBILITY 1. SALTING OUT
MOLECULAR SIZE 1. DIALYSIS
2. GEL FILTRATON
CHROMATOGRAPHY
IONIC CHARGE 1. ION-EXCHANGE
CHROMATOGRAPHY
2. GEL ELETROPHORESIS
3. ISOELECTRIC FOCUSING
BINDING SPECIICITY 1. AFFINITY
CHROMATOGRAPHY

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protei purification slide share mani upadhyay

  • 1. NAME MANI UPADHYAY ROLL NO 2102977 CLASS Bsc(ABS)IV SEM SUB CODE BOM 305 SUBMITTED TO DR.ANURADHA PATEL
  • 3. Proteins are very large molecules composed of basic units called amino acids. Proteins contain carbon, hydrogen, oxygen, nitrogen, and sulphur. Protein molecules are large, complex molecules formed by one or more twisted and folded strands of amino acids WHAT IS PROTEIN
  • 4. WHYWE NEEDTOPURIFY PROTEIN? a.TO IDENTIFY STRUCTURE AND FUNCTION OF PROTEIN OF INTEREST b.TO STUDY PROTEIN REGULATION AND PROTEIN- INTERACTIONS c.TO PRODUCE ANTIBODIES d.PROTEINS ARE USED IN ASSAYS e.PERFORM STRUCTURAL ANLYSIS BY X-RAY CRYSTALLOGRAPHY
  • 5. Proteinprecipitation The main purpose of protein precipitation is to separate the protein from the solution either to eliminate interferences or to purify them. Hydration shell plays a very important role in protein folding and function and is the target of all protein precipitation methods .It is the surrounding water that is formed around proteins, also referred to as “protein hydration” 1. Acid precipitation - This method relies on the changes of a solution pH. Every protein has a defined isoelectronic point or pI value, little changes in a medium pH have an impact on the protein structure. Adding acids to the solution, lowers the pH and leads to positively charging the protein In an aqueous solution the hydration sphere surrounding a protein is disrupted; occurred imbalance in structure leads to precipitation. Most popular acid used in this method is TCA (Trichloroacetic Acid),
  • 6. 2. Salting precipitation - Adding the salt ions into the solution cause the restriction of the available water molecules for the proteins, which leads to destruction of the hydrogen bonds. The interaction between proteins is stronger than between the protein and the available water molecules which causes the protein aggregation and precipitation. Examples are zinc sulphate and ammonium sulphate. This method is widely used for initial fractioning of different proteins, based on their solubility. 3. Alcohol precipitation - Organic solvents can be also used. Adding alcohol to the solution reduces the hydration of the protein, by removing water and surrounding proteins which leads to aggregation and precipitation. To preserve their biological functions, it is
  • 7. CHROMATOGRAPHY In this process, we apply the mixture to be separated on a stationary phase (solid or liquid) and a pure solvent such as water or any gas is allowed to move slowly over the stationary phase, carrying the components separately as per their solubility in the pure solvent. TYPES OF CHROMATOGRAPHY 1. Thin Layer Chromatography In the process of thin-layer chromatography (TLC), the mixture of substances is separated into its components with the help of a glass plate coated with a very thin layer of adsorbent, such as silica gel and alumina, as shown in the figure below. The plate used for this process is known as chrome plate. The solution of the mixture to be separated is applied as a small spot at a distance of 2 cm above one end of the plate. The plate is then placed in a closed jar containing a fluid termed as an eluant, which then rises up the plate carrying different components of the mixture to different heights.
  • 8. 2. Column Chromatography Column chromatography is the technique used to separate the components of a mixture using a column of suitable adsorbent packed in a glass tube, as shown in the figure below. The mixture is placed on the top of the column, and an appropriate eluant is made to flow down the column slowly. Depending upon the degree of adsorption of the components on the wall adsorbent column, the separation of the components takes place. The component with the highest absorptivity is retained at the top, while the other flow down to different heights accordingly.
  • 9. DIALYSIS ‘Dialysis’ is a process of separating PROTEIN molecules or other molecules in particular solutions with the help of a membrane that is semipermeable known as a dialysis tube. It is also used in the studies of drugs and electrophoresis. In dialysis a semipermeable membrane is used to separate small molecules and protein based upon their size. A dialysis bag made of a semipermeable membrane (cellulose) and has small pores. The bag is filled with a concentrated solution containing proteins. Molecules that are small enough to pass through the pores of the membrane diffuse out of the bag into the buffer solution, or dialysate. Dialysis is sometimes used to change buffers. The molecules go from an area of high concentration to low concentration. When the level of concentration is equal between the bag and the buffer, there is no more net movement of molecules. The bag is taken out and inserted into another buffer, causing the concentration to be higher in the bag relative to the buffer. This causes more diffusion of molecules. This process is repeated several times to ensure that all or most of the unwanted small molecules are removed (usually done overnight). In general, dialysis is not a means of separating proteins, but is a method used to remove small molecules such as salts. At equilibrium, larger molecules that are unable to pass through the membrane remain inside the dialysis bag while much of the small molecules have diffused out.
  • 10. Lyophilization If the solution doesn't contain any other soluble component than the protein in question the protein can be lyophilized (dried). This is commonly done after an HPLC run. This simply removes all volatile components, leaving the proteins behind.
  • 11. Electrophoresis THIS TECHNIQUE SEPARTES PROTEINS BASED ON SIZE UNDER AN ELECTRIC FIELD AND CAN BE USED FOR FURTHER PURIFICATION AFTER CHROMATOGRAPHY STEPS UNLESS RESOLUTION OF CLOSELY REALTED SPECIES IS REQUIRED
  • 12. 1. PAPER ELECTROPHORISIS PAPER ELECTROPHORISIS IS ONE OF THE TYPE OF ZONE ELECTROPHORESIS PROCEDURE . • Using an electric field, molecules (such as DNA) can be made to move through a gel made of agar or polyacrylamide. • The electric field consists of a negative charge at one end which pushes the molecules through the gel, and a positive charge at the other end that pulls the molecules through the gel. • The molecules being sorted are dispensed into a well in the gel material. • The gel is placed in an electrophoresis chamber, which is then connected to a power source . • When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. • The different sized molecules form distinct bands on the gel.
  • 13.
  • 14. WHERE DO WE GET PROTEINS 1. WHOLE ORGANISM 2. ORGANS OR TISSUE 3. EMBRYOS 4. PROTEINS FROM EXPRESSION SYSTEM (GENES CLONED INTO A VECTOR , INSERTED INTO A LIVING ORGANISM WHICH MAKES THE PROTEIN)
  • 15. THE BASIC AIM IN PROTEIN PURIFICATION IS TO ISOLATE ONE PARTICULAR PROTEIN OF INTEREST FROM OTHER CONTAMINATING PROTEINS TO STUDY ITS STRUCTURE AND FUNCTION , INCREASING ITS STABILITY AND LARGE SCALE PRODUCTION .
  • 16. APPLICATIONOF PURIFIED PROTEINS 1.HORMONES – INSULIN , ERYTHROPOIETIN , CLOTTING FACTORS 2.DIAGNOSTICS – LFT , KFT , TFT 3.CELLULAR MARKERS – CANCER 4.IN RECOMBINANT DNA TECHNOLOGY TO LOCATE GENE OF INTEREST 5.VACCINES 6.SUPPLEMENTS
  • 17. ISOLATIONOFPROTEINS 1. METHOD OF ISOLATION OF PROTEINS DIFFRS FROM ONE PROTEIN TO OTHER . 2. PROTEINS CAN BE ISOLATED ON THE BASIS OF FOLLOWING PROPERTIES :- CHACTERISTICS PROCEDURE SOLUBILITY 1. SALTING OUT MOLECULAR SIZE 1. DIALYSIS 2. GEL FILTRATON CHROMATOGRAPHY IONIC CHARGE 1. ION-EXCHANGE CHROMATOGRAPHY 2. GEL ELETROPHORESIS 3. ISOELECTRIC FOCUSING BINDING SPECIICITY 1. AFFINITY CHROMATOGRAPHY