PROTEIN PURIFICATION

AND IDENTIFICATION OF
AMINO ACIDS
AREVALO | CUNANAN | DESAI | GENARO | LEE
BONDOC | DE GUZMAN | ESCOTO | KACHHADIYA | LLANERA
 OBJECTIVES
• At the end of the discussion, the student should be able to:
• Define chromatography
• Identify the different types of protein separation
• Discuss the different types of chromatography
• Discuss the general principles of IEC and Paper chromatography
• Definition
• Procedure
• Application
• Discuss the anticipated results and present obtained results.
• Explain possible reasons and sources of error for the deviation of the obtained results from the
anticipated results
• Discuss a journal on IEC
INTRODUCTION
 SIGNIFICANCE OF PROTEIN SEPARATION

• Highly purified protein is essential for the detailed examination of its

amino acid sequence, cell contain thousands of different protein, each
in widely varying amount.

• Classical methods for separating proteins according to their

properties:
• size
• charge
• binding properties

• It is important for the characterization of the function, structure and

interaction of the protein of interest.
• Source: Tissue or microbial cells and body fluids

• Steps (in any protein purification procedure)

1. Break open cells to release their proteins in a crude extract

2. Differential centrifugation

• Subcellular functions

• Specific organelle
TYPES OF PROTEIN
SEPARATION
 TYPES OF PROTEIN SEPARATION
• According to Size:
• Dialysis
• Ultracentrifugation

• According to Charge:
• Electrophoresis

• According to Solubility:
• Salting out
• Isoelectric Precipitation
 DIALYSIS
ACCORDING TO SIZE

• Separates proteins from small solutes

• The partially purified extract is place in a bag with a
semi-permeable membrane
• Membrane allows exchange of salt and buffer, not
proteins
– When bag is suspended in a much larger volume of buffered
solution
 ULTRACENTRIFUGATION
ACCORDING TO SIZE

• CENTRIFUGE - to allow substances to sediment through

gravity by allowing the substance to rotate at high speeds.
• Only when they are subjected to enormous accelerations will
the sedimentation behavior of macromolecules begin to
resemble that of sand grains.
 ELECTROPHORESIS
ACCORDING TO CHARGE

• Migration of charged solutes or particles in an electric field.

• Electrophoretogram is the result of zone electrophoresis and consist of

sharply separated zones of a macromolecule.
• Carried out in gels made up of the cross-linked polymer polyacrylamide.

• Types of Electrophoresis:
• SDS – PAGE
• Isoelectric focusing
• Two-dimensional electrophoresis (combination of SDS-PAGE +
Isoelectric focusing)
 SALTING OUT
ACCORDING TO SOLUBILITY

• Salts may precipitate proteins

• Precipitated proteins are removed from those

remaining in solution by low-speed centrifugation

• Ammonium sulfate is particularly effective and is

often used to salt out proteins.
 ISOELECTRIC PRECIPITATION
ACCORDING TO SOLUBILITY

• Isoelectric point (pI)

• No charge = no electrostatic repulsion
• Thus, protein tend to aggregate and precipitate
CHROMATOGRAPHY
 CHROMATOGRAPHY
• Mixture of substances is fractionated by dissolving it in a
liquid or gaseous fluid and percolated through a column of
porous solid matrix.
• Method of separating the constituents of a solution based on
one or more of its chemical properties:
• Size (size exclusion chromatography)
• Charge (ion-exchange chromatography)
• Hydrophobicity (hydrophobic interaction chromatography)
• Ability to bind a specific ligand (affinity chromatography)
 CHROMATOGRAPHY
• All chromatographic methods require:
• Stationary phase – static part
• Mobile phase – moving part
 TYPES OF
CHROMATOGRAPHY
 TYPES OF CHROMATOGRAPHY
1. Column Chromatography

2. Size Exclusion Chromatography

3. Affinity Chromatography

4. Hydrophobic Interaction Chromatography

5. Thin Layer Chromatography

6. High Performance Liquid Chromatography

7. Ion Exchange Chromatography

8. Paper Chromatography
 COLUMN CHROMATOGRAPHY
• A porous solid (stationary
phase) material is placed in
a column and a buffered
solution migrates through it
• Protein (Mobile Phase)
• Individual proteins migrate
faster or more slowly
through the column
depending on their
properties
 SIZE EXCLUSION CHROMATOGRAPHY

• “Gel filtration”
• Separation according to
size
• Large proteins emerge
from the column sooner
than the small ones
• Solid phase
– Cross-linked polymer
beads with engineered
pores or cavities
 AFFINITY CHROMATOGRAPHY

• Based on binding affinity

• “Ligand”
− Beads in the column
with covalently
charged groups

• Protein with affinity for

this particular chemical
group will bind to the
beads in the column
 HYDROPHOBIC INTERACTION CHROMATOGRAPHY

• Separate proteins based on their tendency to associate with a stationary

phase matrix coated with hydrophobic groups (phenyl Sepharose,
octyl Sephadex)

• Proteins with exposed hydrophobic surfaces adhere to the matrix via

hydrophobic interactions that are enhanced by employing a mobile phase
of high ionic strength.

• Polarity of the mobile phase is decreased by gradually lowering its salt

concentration.

• Addition of ethanol or glycerol = decrease polarity and further weaken

hydrophobic interactions
 THIN LAYER CHROMATOGRAPHY

• Similar to paper chromatography

• Stationary phase: thin layer of a solid (alumina or silica)
supported on an inert base (glass, aluminum foil, or
insoluble plastic)
 HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY

• Makes use of high pressure pumps

– Speed the movement of protein molecules down the column

• By reducing the transit time on the column, HPLC can limit

diffusional spreading of protein bands and thus greatly
improve resolution.
 ION EXCHANGE
CHROMATOGRAPHY
 ION EXCHANGE CHROMATOGRAPHY
DEFINITION

• Differences in the sign

and magnitude of the
net electric charge of
proteins at a given pH

• Resin (Column Matrix)

– Contains bound
charged groups
– Cationic exchangers
– Anion exchangers
 ION EXCHANGE CHROMATOGRAPHY
DEFINITION

• Affinity affected by pH
• As the length of the
column increases,
resolution of 2 types of
proteins is improved,
rate of protein flow
usually decreases
• Increased time spent on
the column, decline in
resolution
 ION EXCHANGE CHROMATOGRAPHY
APPLICATION

• Drinking water analysis for pollution and other constituents

• Determination of water chemistries in aquatic ecosystems
• Determination of sugar and salt content in foods
• Isolation of select proteins.
 ION EXCHANGE CHROMATOGRAPHY
PROCEDURE

A. Preparation of Ion Exchanger

B. Column Packing

C. Column Equilibration

• Sample Application

D. Elution

E. Location of Ninhydrin Positive Material

 ION EXCHANGE CHROMATOGRAPHY
PROCEDURE

A.Preparation of the Ion Exchanger

1. Suspension of cation exchanger in 4.0N HCl. Stir and settle for 5 mins.
2. Decant. Resuspend in HCl. Repeat twice. Filter.
3. Suspension of resin in 1.0N NaOH. Decant. Resuspend in NaOH. Heat for 30
mins.
4. Decant. Resuspension in hot 1.0N NaOH. Heat for 30 mins.
5. Decant and resuspend twice.
6. Cool resin suspension to room temperature. Decant and suspend in water.
7. Wash resin thrice by decantation with distilled water. Filter.
8. Suspend moist resin in 0.1M citrate buffer, pH 3.4.
• DOWEX-50W-X4 was used in the experiment.
 ION EXCHANGE CHROMATOGRAPHY
PROCEDURE

B. Column Packing

• A plug of glass wool or filter paper is placed at the bottom of

the column. Then, buffer suspension was poured into the
column.

• Presence of irregularities and air bubbles = repack column.

 ION EXCHANGE CHROMATOGRAPHY
PROCEDURE

C. Column Equilibration

1. Using a 10mL pipette, gently introduce 0.1M citrate buffer (pH 3.4) until it
reaches 0.0mL mark.
2. Adjust the flow of the eluate such that 1mL of eluate is collected in a time span
of 1 minute.
3. Run down as much of the buffer through the column at this specified flow rate
until the level of the buffer has dropped to 0.1mL above the top of the column.
4. Prepare and label a set of 35 tubes with A and B series.

Sample Application
• Introduction of 2mL of unknown amino acid mixture.
• Allow the amino acid to flow into the column until the level of liquid is at 0.1mL
mark.
 ION EXCHANGE CHROMATOGRAPHY
PROCEDURE

D. Elution
• Three buffers were used:
• Buffer 1: 0.1M citrate buffer, pH 3.4 for test tubes 1a-12a
• Buffer 2: 0.1M citrate buffer, pH 6.2 for test tubes 13a-24a
• Buffer 3: 0.1M sodium carbonate, pH 11 for test tubes 25a-35a

• In each test tube, 3mL fraction was collected

• Remaining buffer was removed at the top of the column using a pipette
• 15mL of 0.2M NaOH was poured and allowed to pass through
• When NaOH reached the 0.2mL mark, 0.1M citrate buffer pH 3.4 was
added.
 ION EXCHANGE CHROMATOGRAPHY
PROCEDURE

E. Location of Ninhydrin Positive Material

• A 1mL aliquot was collected from series A test tubes to series B test
tubes.
• Adjust pH to 5:
• Test tubes with pH 3.4 citrate buffer (1b-12b), add 1N NaOH
• Test tubes with pH 11 sodium carbonate (25b-35b), add 0.5M
HCl
• Addition of 0.1mL ninhydrin reagent to each test tube.
• Test tubes was placed in boiling water bath for 5 mins.
 ION EXCHANGE CHROMATOGRAPHY
PROCEDURE

Anticipated Results:

Test tubes 1A - 12A

• Tubes that act positively to the test are concluded to be negatively charged amino
acid, since they repel the negatively charged resins (cation exchanger). Aspartic acid
and glutamic acid may be present.

Test tubes 13A - 24A

• These tubes that acted positively are concluded to be neutral amino acids since the
increase in pH gradient weakens their interaction with the resin

Test tubes 25A - 35A

• Amino acids present are concluded to be positively charged basic amino acids since
they were strongly bound to the negatively charged resin. They were only eluted by
increasing the ionic strength thereby weakening their charge-charge interactions.
Histidine, Arginine and Lysine may be present.
 ION EXCHANGE CHROMATOGRAPHY
POSSIBLE SOURCES OF ERROR

• Human Error
• Improper pipeting
• Inaccurate buffer measurements
• Insufficient addition of unknown
• Inaccurate flow rate (may be too fast or too slow)

• Experimental Error
• Contamination of reagents
• Improper buffer concentration
• Insufficient protein concentration
 ION EXCHANGE CHROMATOGRAPHY
POSSIBLE SOURCES OF ERROR

• Instrumental Error
• Malfunction of the cation exchanger
• Dirty glasswares and equipments
• Drying of resin
• Vacuum pressure is too high
 PAPER
CHROMATOGRAPHY
 PAPER CHROMATOGRAPHY
DEFINITION

• Works by using strips of filter paper as stationary phase

• Involves partition results of differences in the distribution of solutes

between two phase is water supported by the cellulose molecules of a
paper strip and the mobile phase is a mixture of one or more organic
solvents and water.

• Physical characteristics that determine separation:

• Rate of diffusion
• Solubility of the solute
• Nature of the solvent

• Application: Color pigment separation

 PAPER CHROMATOGRAPHY
PROCEDURE

A. Preparation of the Paper Strip

B. Spotting the Paper

C. Development of the Chromatogram (Descending

Technique)

D. Identification of the Amino Acid

 PAPER CHROMATOGRAPHY
PROCEDURE

A. Preparation of the Paper Strip

1. In a 500mL beaker place BUTANOL: ACETIC ACID:

WATER in 4:1:5 proportions. Cover and stand overnight
for equilibration

2. Draw lines at 10, 5.7, 2.2 cm across the 10 inch width of

the Whatman No.1 filter paper. Fold at the 5.7 & 2.2 cm
lines.

3. Lay out series of points on the 10cm line, 1 inch apart.

 PAPER CHROMATOGRAPHY
PROCEDURE

B. Spotting the Paper

1. Standard amino acids

• Prepare 1mg/mL solution of each amino acid and spot the origin
point 2-5x using a cappilet on the same spot on the paper. Keep each
spot within 7mm size.

2. Collect 1mL aliquot each of test tubes 1a-35a.

3. Adjust pH to 5 of test tubes 1a-12a aliquot by adding 1N NaOH and of

test tubes 25a-35a aliquot by adding 0.5M HCl.

4. Spot the origin points with sample from each 10x on the same spot if
Ninhydrin (+) survey tubes were dark; if lighter, spot 15x
 PAPER CHROMATOGRAPHY
PROCEDURE

C. Development of the Chromatogram (Descending Technique)

1. Hang the paper in the chromatography jar.

2. Pour the solvent mixture in trough on top of the jar.

3. Allow solvent to run down the paper until it reaches about 2

inches from the bottom of the sheet (about 13 hrs).

4. Mark the solvent front with pencil and dry the paper.
 PAPER CHROMATOGRAPHY
PROCEDURE

D. Identification of the Amino Acids

1. Spray or dip the paper in Ninhydrin solution [0.2%].

2. Dry the paper in the oven for 3 mins at 95C.

3. Immediately locate and encircle spots with pencil.

4. Calculate the Rf values of the unknown and compare

with the standard amino acids.
 PAPER CHROMATOGRAPHY
PROCEDURE

Anticipated Results:

• Positive tubes from the IEC experiment were used to be able to

determine the amino acids and compare to the 3 standard
amino acids.

• Distance travelled by solute relative to the solvent is the

retention factor which is the distance travelled by solute
divided by the distance travelled by solvent.
 PAPER CHROMATOGRAPHY
POSSIBLE SOURCES OF ERROR

• During application of spots

• Applying too much concentrated solution that may have diffused thereby,
leading to poor separation.
• Inadequate spaces between the spots.

• In development
• Improper adjustment of the paper in the tank leads to this error so the paper
should be placed vertically stable and at the right distance.
• Inaccurate computation of Rf values.

• In detection
• Spraying method can greatly affect the final results leading to inaccurate
measurements of distance.
DATA & RESULTS
 ION EXCHANGE CHROMATOGRAPHY
GROUPS 1 & 2
NINHYDRIN NINHYDRIN NINHYDRIN
BUFFER 1 RESULT BUFFER 2 BUFFER 3
RESULT RESULT

1A +4 13A +2 25A +2
2A +2 14A +2 26A +2
3A +4 15A +2 27A +2
4A +4 16A +4 28A +4
5A +2 17A +2 29A +4
6A +2 18A +4 30A +2
7A +2 19A +4 31A +2
8A +2 20A +2 32A +2
9A +2 21A +2 33A +2
10A +2 22A +2 34A +2
11A +2 23A +2 35A +4
12A +2 24A +2
 ION EXCHANGE CHROMATOGRAPHY
GROUPS 3 & 4
NINHYDRIN NINHYDRIN NINHYDRIN
BUFFER 1 RESULT BUFFER 2 RESULT BUFFER 3 RESULT

1A +1 13A - 25A +2
2A +4 14A +1 26A +2
3A +1 15A +4 27A +4
4A +1 16A +4 28A +3
5A +1 17A +3 29A +3
6A +2 18A +3 30A +2
7A +2 19A +3 31A +2
8A +2 20A +2 32A +2
9A +2 21A +2 33A +2
10A +2 22A +2 34A +2
11A +3 23A +2 35A -
12A +3 24A +2
 ION EXCHANGE CHROMATOGRAPHY
GROUPS 5 & 6
NINHYDRIN NINHYDRIN NINHYDRIN
BUFFER 1 RESULT BUFFER 2 RESULT BUFFER 3 RESULT

1A +1 13A +1 25A +4
2A +2 14A +2 26A +4
3A +4 15A +3 27A +4
4A +4 16A +4 28A +3
5A +4 17A +4 29A +3
6A +3 18A +4 30A +1
7A +1 19A +3 31A +2
8A +2 20A +3 32A +2
9A +1 21A +3 33A +1
10A +2 22A +3 34A +3
11A +2 23A +3 35A +3
12A +1 24A +3
 ION EXCHANGE CHROMATOGRAPHY
GROUPS 7 & 8
NINHYDRIN NINHYDRIN NINHYDRIN
BUFFER 1 RESULT BUFFER 2 RESULT BUFFER 3 RESULT

1A +4 13A +0.5 25A +3

2A +4 14A +4 26A +4
3A +4 15A +4 27A +4
4A +3 16A +4 28A +3
5A +2 17A +2 29A +4
6A +3 18A +2 30A +1
7A +2 19A +2 31A +2
8A +2 20A +1 32A +2
9A +1 21A +2 33A +3
10A +3 22A +2 34A -
11A +1 23A +1 35A -
12A +1 24A +1
 ION EXCHANGE CHROMATOGRAPHY
GROUPS 9 & 10
NINHYDRIN NINHYDRIN NINHYDRIN
BUFFER 1 RESULT BUFFER 2 RESULT BUFFER 3 RESULT

1A +4 13A - 25A +3
2A +4 14A - 26A +3
3A +4 15A +2 27A +3
4A +3 16A +2 28A +4
5A +3 17A +4 29A +3
6A +3 18A +4 30A +2
7A +3 19A +4 31A +2
8A +3 20A +4 32A +3
9A +1 21A +3 33A +3
10A +1 22A +3 34A +2
11A +1 23A +3 35A +1
12A +1 24A +3
 ION-EXCHANGE
CHROMATOGRAPHY RESULTS
GROUPS 1&2 GROUPS 3&4 GROUPS 5&6 GROUPS 7&8 GROUPS 9&10
BUFFER 1

1A +4 +1 +1 +4 +4
2A +2 +4 +2 +4 +4
3A +4 +1 +4 +4 +4
4A +4 +1 +4 +3 +3
5A +2 +1 +4 +2 +3
6A +2 +2 +3 +3 +3
7A +2 +2 +1 +2 +3
8A +2 +2 +2 +2 +3
9A +2 +2 +1 +1 +1
10A +2 +2 +2 +3 +1
11A +2 +3 +2 +1 +1
12A +2 +3 +1 +1 +1
 ION-EXCHANGE
CHROMATOGRAPHY RESULTS
GROUPS 1&2 GROUPS 3&4 GROUPS 5&6 GROUPS 7&8 GROUPS 9&10
BUFFER 2

13A +2 - +1 +1 -
14A +2 +1 +2 +4 -
15A +2 +4 +3 +4 +2
16A +4 +4 +4 +4 +2
17A +2 +3 +4 +2 +4
18A +4 +3 +4 +2 +4
19A +4 +3 +3 +2 +4
20A +2 +2 +3 +1 +4
21A +2 +2 +3 +2 +3
22A +2 +2 +3 +2 +3
23A +2 +2 +3 +1 +3
24A +2 +2 +3 +1 +3
 ION-EXCHANGE
CHROMATOGRAPHY RESULTS
BUFFER 3 GROUPS 1&2 GROUPS 3&4 GROUPS 5&6 GROUPS 7&8 GROUPS 9&10

25A +2 +2 +4 +3 +3
26A +2 +2 +4 +4 +3
27A +2 +4 +4 +4 +3
28A +4 +3 +3 +3 +4
29A +4 +3 +3 +4 +3
30A +2 +2 +1 +1 +2
31A +2 +2 +2 +2 +2
32A +2 +2 +2 +2 +3
33A +2 +2 +1 +3 +3
34A +2 +2 +3 0 +2
35A +4 - +3 0 +1
 PAPER CHROMATOGRAPHY
GROUPS 1 & 2
Distance
Test tube # travelled
Computed Rf Amino Acid

1 3.2 cm 0.07 Lysine

Glutamic
2 6.7 cm 0.16
Acid
Aspartic
3 5.2 cm 0.13
Acid

3a 4.8 cm 0.12 Arginine

Aspartic
4a 5.1 cm 0.13
Acid
18a 4.9 cm 0.12 Arginine

19a 4.3 cm 0.11 Arginine

28a 4.0 cm 0.10 Lysine

29a 2.7 cm 0.07 Histidine

 PAPER CHROMATOGRAPHY
GROUPS 3 & 4
Distance
Test tube # travelled
Computed Rf Amino Acid
Serine /
1 3.1 cm 0.09 Lysine
Alanine /
2 7.6 cm 0.23 Threonine

3 6 cm 0.18 Glycine

Glutamic
11a 5 cm 0.15 Acid
Glutamic
12a 5 cm 0.15 Acid

15a 2.6 cm 0.08 Histidine

17a 2.5 cm 0.08 Histidine

27a 1.9 cm 0.08 Histidine

29a 2.6 cm 0.09 Histidine

 PAPER CHROMATOGRAPHY
GROUPS 5 & 6
Test tube # Distance travelled Computed Rf Amino Acid

1 3.4 0.10 Lysine

2 7 0.17 Glycine
5.9 0.16 Glutamic
3 acid

5.1 0.13 Aspartic

4a acid
5.6 0.13 Aspartic
5a acid
2.8 0.07 Histidine
17a
2.8 0.07 Histidine
18a
2.8 0.07 Histidine
26a

27a 3.5 0.10 Lysine

 PAPER CHROMATOGRAPHY
GROUPS 7 & 8
Distance
Test tube # travelled
Computed Rf Amino Acid

1 3.4 cm 0.084 Histidine

2 5.3 cm 0.13 Glutamine

3 5.8 cm 0.14 Lysine

2a 5.1 cm 0.13 Glutamine

3a 4.6 cm 0.11 Histidine

15a 2.0 cm 0.05 Histidine

16a 2.2 cm 0.05 Histidine

26a 2.2 cm 0.05 Histidine

27a 2.4 cm 0.05 Histidine

 PAPER CHROMATOGRAPHY
GROUPS 9 & 10
Distance
Test tube # travelled
Computed Rf Amino Acid

1 3.2 cm 0.08 Lysine

2 7.5 cm 0.18 Glycine

Glutamic
3 6 cm 0.15
Acid

Aspartic
2a 5.6 cm 0.14
Acid
Aspartic
3a 5.5 cm 0.14
Acid
18a 2.8 cm 0.07 Histidine

19a 2.7 cm 0.07 Histidine

26a 2.7 cm 0.07 Histidine

27a 2.7 cm 0.07 Histidine

 THE ALLERGENS OF SCHISTOSOMA
MANSONI
II. FURTHER SEPARATION BY
SEPHADEX G-200 AND ION-EXCHANGE
CHROMATOGRAPHY

W. G. HARRIS
 One of the most consistent features of helminth immunity is the
ability of these parasites to stimulate a high titre reagin response. Such
antibody has been detected in a wide range of helminth host-parasite
systems, including schistosome infections of mice, rats, rabbits,
monkeys and man. This powerful immunological effector mechanism
must, therefore, play a significant role in the ultimate host-parasite
relationship, and a study of parasite allergens should yield information
relevant to the fundamental understanding of this relationship.
 SCHISTOSOMIASIS
• Remains one of the most prevalent neglected tropical diseases

• Considered as a major public health problem in about 77 developing

countries in tropics and subtropics

• It is estimated that over 240 million people are infected, with about 700
million people worldwide at risk of infection

• Prevalence and morbidity is highest among school children, adolescents,

and young adults

• The overall prevalence of Schistosomiasis was 17.8% with 8.9% infection

with S. mansoni and 8.3% with S. haematobium
 OBJECTIVE

To discover the significant use of purified antigens

in diagnosis of Schistosomiasis
 MATERIALS AND METHODS
• The parasite: Schistosoma mansoni

• Preparation of antigens

• Separation of Sephadex G-200

• Separation on DEAE-cellulose
– Fraction SAIGl/Ul G-200 high molecular weight
acid insoluble fraction was further separated by ion-
exchange chromatography on DEAE-cellulose
 MATERIALS AND METHODS
• Separation on CM-cellulose
– Fraction SASGI/Ul-the Sephadex G-200 high
molecular weight acid-soluble peak and fraction
SASG3-the Sephadex G- 100 medium molecular
weight peak-were further separated by ion-exchange
chromatography on CM-cellulose

• Assay of allergic activity

– each antigen fraction in the allergen-reagin axis was
determined by modified Prausnitz-Kustner (P-K)
type assay in rats
 RESULTS

• Fraction SASGl/U1 was the most active, with

SASGI /U2 and U3 showing slightly lower activity

• The principal allergen of the acid-soluble, high

molecular weight group was fraction SASG1/Ul

• The principal allergen of the acid-insoluble, high

molecular weight group was fraction SAIGl/U2
 RESULTS
• CM-cellulose
– revealed specific activity in a range of molecular groups
– Neither of these fractions possessed any non-specific activity
• DEAE-cellulose
– Allergic activity was detected in three principal molecular
groups
• Direct separation of fraction SASG3 on CM-cellulose
– at least three allergens were present, one eluting with starting
buffer, one at about 0.2 salt and the third at about 0.4M salt.
 REFERENCE
• Nelson, D., Cox, M. (2013). Lehninger Principles of Biochemistry, 6th edition

• Devlin, T. Textbook of Biochemistry with Clinical Correlations, 4th edition

• Harris, W.G. The Allergens of Schistosoma mansoni

• Rodwell, V.W, et.al. (2015). Harper’s Illustrated Biochemistry, 30th edition