Protein Purification
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Molecular weight
Charge
Solubility
Affinity
Molecular Weight
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Ultracentrifugation
Dialysis
Gel filtration
SDS PAGE
Molecular Weight
• The lab in week 6 and 7 will involve
separating a protein mixture by molecular
weight using 2 methods: gel filtration and
SDS PAGE
• Gel filtration separates by the native
molecular weight
• SDS PAGE separates by the subunit
molecular weight
Gel Filtration
• This method relies on a column of beads
of a specified pore size. This is known as
a molecular sieve.
• Proteins (and other macromolecules)
above a certain cut-off size cannot fit into
the pores and so migrate down the outside
of the beads. They will elute first.
• Smaller molecules below the cut-off can
permeate the pores and so take longer to
travel down the column.
An elution profile
Heavier
Lighter
• Gel-filtration of the
protein mixture. 1.2 ml
of protein mixture (10
mg/ml) was loaded onto a
25 cm X 2.5 cm diam.
Sephadex G-50 column
equilibrated with buffer
(50 mM Tris HCl, pH 7.5).
The column was eluted
with buffer at ~1 ml/min,
collecting 2.5 ml fractions.
The absorbance of each
fraction was measured at
280 nm and 410 nm.
SDS PAGE
• This technique involves loading a sample
of your mixture onto a polyacrylamide gel
(PAGE). Polyacrylamide works like
agarose except the matrix has smaller
pores and so polyacrylamide gels
separate smaller molecules (like proteins).
Agarose is used for much larger molecules
such as DNA and RNA.
SDS PAGE
• Unlike DNA and RNA proteins do not have
a nice constant charge to mass ratio and
can have any charge at a given pH,
depending on their sequence, hence pI.
• To overcome this problem proteins are
coated with a detergent, SDS, which
makes them negatively charged.
• They then separate by molecular weight.
SDS PAGE
• They then separate by molecular weight.
• The SDS will disrupt the secondary,
tertiary and quaternary structure so the
subunits will separate. For this reason
SDS-PAGE separates by subunit
molecular weight.
SDS-PAGE
1.
2.
3.
4.
5.
6.
Catalase, cytochrome C,
a-lactalbumin
Hemoglobin, Cytochrome C,
a-lactalbumin
BSA, cytochrome C,
a-lactalbumin
Hemoglobin, myoglobin,
a-lactalbumin
Ferritin, cytochrome C,
a-lactalbumin
Ferritin, myoglobin,
lighter
a-lactalbumin
1
2
3
4
5
6
Your Task
• Each pair will be given a mixture of three
proteins. This mixture will be unique to your
group. Your mixture will contain between 4 and
8 mg of any three of the following proteins:
Myoglobin, Haemoglobin, Cytochrome c,
a-lactalbumin, Ribonuclease, Bovine Serum
Albumin, Ferritin and Catalase. It is your task to
separate and identify these three proteins.
Clues to help you
• Your mixture of three proteins will either contain
2 heavy and 1 light protein or 1 heavy and 2 light
proteins. For the purposes of this experiment, a
heavy protein is defined as one with a molecular
weight of over 50,000 and a light protein is one
with a molecular weight of less than 50,000.
The two smaller/heavier proteins in the mixture
have pI’s that differ by at least 2 pH units. These
can be separated by ion exchange
chromatography at pH 7.5.
Hint…..
• If you can separate 2 proteins by ion
exchange chromatography at pH 7.5 then
the 2 proteins must have pIs on either side
of 7.5 so they are opposite charges at PpH
7.5.
• Knowing information about the possible
proteins at the back of the notes for this
lab session what can you conclude before
coming to class?
Charge
• Ion Exchange Chromatography
• Native gel electrophoresis
• Isoelectric focusing
Charges on proteins
• Different proteins have different native
charges.
• The overall charge on a protein will
depend on:
– The sequence
– The pH
Determining the pI of a protein
• It can be predicted from the difference
between the sum of the acidic side chains
(asp + glu) and the sum of the basic side
chains (lys + arg + his).
• It is determined experimentally by
techniques such as isoelectric focusing.
The protein is placed in a pH gradient and
subjected to an electric field. The protein
moves to its pI.
Determining the pI of a protein
• Those proteins with more acidic residues
will have a lower pI
• Those proteins with more basic residues
will have a higher pI.
Estimating the charge of a protein
• What we really want to know is the charge
of a protein at a particular pH, like 7.
• How do we use pI data to predict the
charge of our protein?
• Acidic residues lower the pI
• Basic residues raise the pI.
Estimating the charge of a protein
[H+]
pI ~5
[OH-]
Protein becomes
increasingly +ve
[H+]
[OH-]
Protein becomes increasingly -ve
Estimating the charge of a protein
At pH 3 the
protein will
be +ve
[H+]
pH ~3
[OH-]
Protein becomes
increasingly +ve
pI ~5
[H+]
[OH-]
Protein becomes increasingly -ve
Estimating the charge of a protein
At pH 7
the protein
will be -ve
[H+]
pI ~5
[OH-]
Protein becomes
increasingly +ve
pH ~7
[H+]
[OH-]
Protein becomes increasingly -ve
At a particular pH..
• If the pH of the environment is below
(more acidic >[H+]) the pI then the protein
will be positive (+ve)
• If the pH of the environment is above
(more basic >[OH-]) the pI then the protein
will be negative (-ve).
Ion Exchange Chromatography
• If the column is positively charged i.e.
DEAE then….
• Proteins with pIs l < the pH of the buffer
will be negatively charged and bind to the
column.
• Proteins with pIs > the pH of the buffer will
be positively charged and will not bind to
the column but elute.
Ion Exchange Chromatography
• If the column is negatively charged
charged i.e. carboxymethyl then….
• Proteins with pIs l < the pH of the buffer
will be negatively charged and not bind to
the column but elute.
• Proteins with pIs > the pH of the buffer will
be positively charged and will bind to the
column.
Native Gel Electrophoresis
• Proteins with pIs l < the pH of the buffer
will be negatively charged and will move to
the anode (+ve), the red electrode!!
• Proteins with pIs > the pH of the buffer will
be positively charged and will move to the
cathode (-ve), the black electrode!!
Isoelectric Focusing
• A pH gradient is set up along the length of
the gel
• An electric field is applied
• Proteins move to the point where they no
longer have a charge i.e. their pI
• Used as the first dimension of 2D gel
electrophoresis
Proteomics
• A combination of isoelectric focusing (first
dimension) and SDS PAGE (second
dimension) can separate the complete
proteome of a cell!
• You produce spots which can be cut out
and analysed by mass spectrometry.
• Compare to libraries of proteins
2D gel electrophoresis
pH
3
10
High mol. Wt.
SDS PAGE
Low mol. Wt.
Affinity
• Exploited with cloning
• His-tagged proteins purified on Nickel
columns.
• GST fusion proteins purified on glutathione
columns.