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Protein Chromatography: Ion Exchange Chromatography Aims

Ion exchange chromatography is used to separate charged proteins by binding them to ion exchange resin columns based on their charge. The document describes conducting ion exchange chromatography using a crude protein extract to isolate and purify proteins. Key steps include equilibrating the column, loading the sample, washing unbound proteins, creating a salt gradient to elute bound proteins in fractions, analyzing the fractions using protein gel electrophoresis and a protein assay to identify purified proteins.

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157 views4 pages

Protein Chromatography: Ion Exchange Chromatography Aims

Ion exchange chromatography is used to separate charged proteins by binding them to ion exchange resin columns based on their charge. The document describes conducting ion exchange chromatography using a crude protein extract to isolate and purify proteins. Key steps include equilibrating the column, loading the sample, washing unbound proteins, creating a salt gradient to elute bound proteins in fractions, analyzing the fractions using protein gel electrophoresis and a protein assay to identify purified proteins.

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kapilphysio
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PROTEIN CHROMATOGRAPHY:

Ion Exchange Chromatography


Aims
1. Learn the principle of ion exchange chromatography.
2. Understand the immobilization of protein on ionic charged columns.
3. Learn factors influencing binding and elution of proteins during ion exchange
chromatography.
4. A hands-on ion- exchange chromatography lab activity.
Background
A key step in proteomics, the study of proteins function and structure, is the purification
of proteins. The ability to isolate and purify specific proteins is an essential feature of
modern biochemistry as it allows scientist to study proteins in isolation from other
proteins, which greatly aids the understanding of a particular protein's function.
Unfortunately, there is no single ideal protein purification procedure and often the
purification of a protein involves several techniques. The main idea behind protein
purification is to select the best technique to isolate a protein of interest, based on
differences in their physical properties from other unwanted proteins. One general
separating technique, with many different approaches, is chromatography.
Ion exchange chromatography is used to separate charged molecules, including proteins,
from complex biological samples. Charged substances are separated by column
chromatography with resins that carry charged ionic groups. Biomolecules such as
proteins, with an opposite charge will bind to the resins. The ionic groups of the columns
are covalently bound to a gel matrix and are protected by small concentrations of counter
ions that are present in buffer. When a sample is added to the column, an exchange with
the weakly bound counter ions takes place and charged molecules bind to the solid
support.
Proteins contain the regions of charged groups on their surface that are formed by the side
groups of charged aminoacids, the -amino and -carboxyl termini of the polypeptide
chains, and other interacting groups. These charged groups are available for interaction
and exchange with ionic groups of the ion exchange columns.
Proteins are multivalent anions or cations and the protonation (adding of the proton) of
protein molecules changes with changes in pH. Under strongly acidic pH conditions, all
proteins are present as cations as the results of the suppression of the dissociation of the
carboxyl groups and protonation of the amino groups. At pH values above12, proteins are
present as anions due to the amino group being a free base and the carboxy group is

dissociated. Depending on the total charge of a protein and the wide range of the ion
exchange columns available it is simple to bind proteins of interest to a corresponding
charged stationary phase for purification.
During the practical application of ion exchange chromatography it is important to use
pH values that ensure the ionic exchange resins are in an ionized state and the proteins
contain an excess of positive or negative charges , i.e. they are not near their pI
(isoelectric point) value, the net charge is zero.

Increasing the salt concentration results in the shielding of the charges on the protein's
surface and effective binding to an exchanger is inhibited. Also changing the pH of the
binding buffer changes the ionization of the protein charged groups and results in the
breaking of the interaction with the ion exchange columns. As a results, proteins
immobilized on an ion exchange column can be eluted either by increasing the salt
concentration or by altering the pH of the binding buffer, or a combination of the two.
This lab activity involves using a crude protein extract and running ion exchange
chromatography for isolation of proteins from the protein extract .

Materials used:
1.
2.
3.
4.
5.
6.
7.

1 Anionic Chromatography Column


1 vial freshly prepared Tissue extract
10 ml Equilibration Buffer
1ml Elution buffer
12ml CB Protein Assay Reagent
110l 2X Sample Loading Buffer
Reagents for 1 SDS polyacrylamide gel for protein electrophoresis

Procedure:
Always wear gloves and protecting clothing throughout the whole experiment.

1. Ion exchange chromatography


1. Pour the resolving gel according to the Protein Electrophoresis protocol.
2. Place the Anionic Chromatography column in an upright position in the tube
stand.

3. Open the top cap first and then the bottom cap of the column to prevent air
entering the resins. Allow the buffer to drain out of the column, under gravity ,to a
waste container.
4. Equilibrate the column: Apply 2 volumes (0.5ml each) of Equilibration buffer
(containing 10m M Tris-HCL, pH 7.5). Add 0.5 ml Equilibration buffer, allow the
buffer to drain out, i.e. when it stop dripping , and then apply the second volume.
Add the equilibration buffer slowly to avoid disturbing the resin in the column.
5. Carefully load 100l Tissue extract to the column.
6. Wash the column 3 times (0.5ml each) to remove unbound protein from the
column :Apply 0.5ml Equilibration buffer to the column and let the buffer drain
out into a waste container.
7. Elute the sample using the salt gradient: For preparing a salt gradient elution
buffer mix Equilibration buffer and Elution buffer, which contains a high
concentration of salt as in the table below.
Fraction
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Salt Concentration
(M)
0.02
0.04
0.06
0.10
0.14
0.20
0.30
0.40
0.50
0.60

Equilibration Buffer
(ml)
0.495
0.490
0.485
0.475
0.465
0.450
0.425
0.400
0.375
0.350

Elution Buffer (ml)


0.005
0.010
0.015
0.025
0.035
0.050
0.075
0.100
0.125
0.150

8. Apply 0.5ml of each gradient elution buffer to the column, starting from the
lowest salt concentration buffer
9. Collect 0.5ml fractions as the buffer freely drains into collection tubes. Change to
a fresh tube before you apply next elution buffer. Collect all 10 fractions in 10
separate 1.5ml tubes.
10. Pour stacking gel according to the Protein Electrophoresis protocol.
11. Determine the protein concentration of each fraction by following the instructions
in section 3.

Protein Electrophoresis:
1. Transfer 10l each fraction to a fresh tube.
2. Add 10l 2X Loading buffer and boil for 5 minutes.

3.
4.
5.
6.

Vortex the sample briefly and centrifuge for 30 seconds.


Set up the electrophoresis gel to load your samples.
Load all the protein samples to the lanes.
Run the gel at 100 volts until the blue dye front is 1.0 cm from the bottom of the
gel.
7. Disassembly the gel carefully.
8. Wash the gel twice in distilled water, five minutes each.
9. Remove all free water from the gel.
10. Add 50ml Lab Safe Gel Stain to cover the gel. Gently shake the gel for 60
minutes at room temperature.
Protein bands will start to appear after 10 minutes. Check gels at regular intervals to
see the proteins appear.
11. Decant the Lab Safe Gel Stain and rinse the gel with distilled water. The gel can
be stored in water. Longer destaining, such as overnight, in water will give a
clearer view of the protein bands.

CB Protein Assay:
1. Label 10 tubes and transfer 100l elute from each fraction.
2. Mix the CB Protein Assay Reagent gently by inverting the bottle several times.
To avoid foaming , DO NOT SHAKE THE BOTTLE.
3. Add1ml CB Protein Assay Reagent to each tube and vortex briefly to mix the
content. Incubate the tubes at room temperature for 5 minutes.
4. Transfer 250l from each assay tube to a microtiter plate well. Read the
absorbance at 595nm.
5. Measure the absorbance of the plate and plot a graph of absorbance against the
fraction number.

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