Worksheet: EXPERIMENT 2.6: SEPARATING
MOLECULES - AFFINITY CHROMATOGRAPHY
18 November 2020
09:20
SESSION ORGANISATION
All timings are approximate.
10.00 a.m. There will be a brief introduction by the Academic Lead together with
any course-related announcements, followed by a video demonstrating the
technique and experiment.
10.30 a.m. – 12.00 p.m. Complete worksheet in your groups with help from your
demonstrator: remember that while you are working as a group, each of you
must complete their own worksheet, in your own words.
Aim of the To purify α-lactalbumin from
experiment milk whey, using affinity
chromatography.
To identify the proteins in
cow’s milk whey
To determine the purity of α-
lactalbumin separated from
milk whey
Your learning 1. To understand the
objectives theoretical basis of affinity
chromatography.
2. To gain experience of
experimental planning
3. To use A280 measurements
to follow elution of proteins
from a column
4. To use gel electrophoresis
to analyse samples and
determine the purity of a
protein
INTRODUCTION
,Affinity chromatography allows molecules to be separated on the basis of their
specific biological properties, rather than physical characteristics such as size or
shape. Some proteins bind metal ions, and so for these proteins, metal ions can
be used as a ligand.
In this experiment you will use copper ions (Cu2+) to bind α-lactalbumin, one of
the main proteins from milk whey. Firstly, you need to attach the metal ions to a
suitable support (a matrix of chelating Sepharose). You then apply your sample,
and collect those proteins which do not bind to the column. You will use
imidazole to displace the bound proteins (it does this by competing with the
electron-rich residues for the metal binding sites).
Cow’s milk contains a number of proteins. The most abundant proteins are
caseins (there are several closely related types of casein in cow’s milk), which
are easily precipitated under acid conditions. You see this process occurring
when milk turns sour; acid formed by bacteria in the milk precipitates the
caseins, causing the milk to separate – you see lumps of precipitated casein in
a yellowish fluid, called whey. When the caseins are removed, the whey
contains several proteins. The most abundant are α-lactalbumin, and β-
lactoglobulin. Others e.g. bovine serum albumin and immunoglobulins are
present in smaller amounts. All these proteins are relatively small; the table
below shows the molecular mass of each protein.
Protein Molecular mass (Daltons)
caseins 25,000 – 35,000
α-lactalbumin 14,000
β-lactoglobulin 18,000
In this experiment, you will remove the caseins from a sample of milk,
and use affinity chromatography to purify α-lactalbumin from the
remaining whey.
Monitoring the protein content of column eluates using absorbance
at 280 nm
The proteins under investigation in this experiment are not as easy to detect as
the proteins you have investigated so far, as they do not absorb light in the
, visible region (i.e. they are not coloured). However, because they contain amino
acids with aromatic sidechains, proteins absorb light in the UV region, at 280
nm. Clearly, since different proteins contain different numbers of aromatic amino
acid residues, the molar absorption coefficient (ε280 varies from one protein to
another, so this method is not an accurate way of determining protein
concentration, unless you know the value of ε280 for every protein). However, it
gives a guide to protein concentration (1 mg/ml protein has an absorbance of
approximately 1.0), is quick, and does not destroy any of your sample. Not all
spectrophotometers can produce light in the UV region, and normal plastic
cuvettes cannot be used, as they are not transparent to UV light. Special
cuvettes are provided; they are expensive, so use the same cuvette throughout.
PREPARATION
This practical occupies both sessions this week. Before you come to the lab,
you should read the background materials on Minerva, Portal and VLE, read
through the experimental protocol, and using the information from these
sources, complete the preparation MCQ questions.
Do not complete the following sections of the worksheet until the
session begins.
Watch the video/demonstration and note the key points of the method here.
METHOD
You are provided with the following materials:
¨ Milk powder
¨ 10% acetic acid
¨ Suspension of chelating Sepharose
¨ 1M copper sulphate solution in water
¨ Buffer A; 0.1 M acetate, 0.5 M NaCl pH 4.5
¨ Buffer B; 0.1 M acetate, 0.5 M NaCl, 20 mM imidazole pH 4.5
¨ Bovine serum albumin (BSA) powder
MOLECULES - AFFINITY CHROMATOGRAPHY
18 November 2020
09:20
SESSION ORGANISATION
All timings are approximate.
10.00 a.m. There will be a brief introduction by the Academic Lead together with
any course-related announcements, followed by a video demonstrating the
technique and experiment.
10.30 a.m. – 12.00 p.m. Complete worksheet in your groups with help from your
demonstrator: remember that while you are working as a group, each of you
must complete their own worksheet, in your own words.
Aim of the To purify α-lactalbumin from
experiment milk whey, using affinity
chromatography.
To identify the proteins in
cow’s milk whey
To determine the purity of α-
lactalbumin separated from
milk whey
Your learning 1. To understand the
objectives theoretical basis of affinity
chromatography.
2. To gain experience of
experimental planning
3. To use A280 measurements
to follow elution of proteins
from a column
4. To use gel electrophoresis
to analyse samples and
determine the purity of a
protein
INTRODUCTION
,Affinity chromatography allows molecules to be separated on the basis of their
specific biological properties, rather than physical characteristics such as size or
shape. Some proteins bind metal ions, and so for these proteins, metal ions can
be used as a ligand.
In this experiment you will use copper ions (Cu2+) to bind α-lactalbumin, one of
the main proteins from milk whey. Firstly, you need to attach the metal ions to a
suitable support (a matrix of chelating Sepharose). You then apply your sample,
and collect those proteins which do not bind to the column. You will use
imidazole to displace the bound proteins (it does this by competing with the
electron-rich residues for the metal binding sites).
Cow’s milk contains a number of proteins. The most abundant proteins are
caseins (there are several closely related types of casein in cow’s milk), which
are easily precipitated under acid conditions. You see this process occurring
when milk turns sour; acid formed by bacteria in the milk precipitates the
caseins, causing the milk to separate – you see lumps of precipitated casein in
a yellowish fluid, called whey. When the caseins are removed, the whey
contains several proteins. The most abundant are α-lactalbumin, and β-
lactoglobulin. Others e.g. bovine serum albumin and immunoglobulins are
present in smaller amounts. All these proteins are relatively small; the table
below shows the molecular mass of each protein.
Protein Molecular mass (Daltons)
caseins 25,000 – 35,000
α-lactalbumin 14,000
β-lactoglobulin 18,000
In this experiment, you will remove the caseins from a sample of milk,
and use affinity chromatography to purify α-lactalbumin from the
remaining whey.
Monitoring the protein content of column eluates using absorbance
at 280 nm
The proteins under investigation in this experiment are not as easy to detect as
the proteins you have investigated so far, as they do not absorb light in the
, visible region (i.e. they are not coloured). However, because they contain amino
acids with aromatic sidechains, proteins absorb light in the UV region, at 280
nm. Clearly, since different proteins contain different numbers of aromatic amino
acid residues, the molar absorption coefficient (ε280 varies from one protein to
another, so this method is not an accurate way of determining protein
concentration, unless you know the value of ε280 for every protein). However, it
gives a guide to protein concentration (1 mg/ml protein has an absorbance of
approximately 1.0), is quick, and does not destroy any of your sample. Not all
spectrophotometers can produce light in the UV region, and normal plastic
cuvettes cannot be used, as they are not transparent to UV light. Special
cuvettes are provided; they are expensive, so use the same cuvette throughout.
PREPARATION
This practical occupies both sessions this week. Before you come to the lab,
you should read the background materials on Minerva, Portal and VLE, read
through the experimental protocol, and using the information from these
sources, complete the preparation MCQ questions.
Do not complete the following sections of the worksheet until the
session begins.
Watch the video/demonstration and note the key points of the method here.
METHOD
You are provided with the following materials:
¨ Milk powder
¨ 10% acetic acid
¨ Suspension of chelating Sepharose
¨ 1M copper sulphate solution in water
¨ Buffer A; 0.1 M acetate, 0.5 M NaCl pH 4.5
¨ Buffer B; 0.1 M acetate, 0.5 M NaCl, 20 mM imidazole pH 4.5
¨ Bovine serum albumin (BSA) powder
