biochemistry

1. HBC1011 Biochemistry I
Trimester I, 2018/2019
Lecture 9 – Exploring Protein Part I
Ng Chong Han, PhD
MNAR1010, 06-2523751
chng@mmu.edu.my

2. Overview
Methods to study protein
• Purification & Characterization
– Protein Source
– Identification Assay
– Homogenization
– Differential centrifugation
– Salting out
– Dialysis
– Chromatography
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3. Protein methods
• Techniques used to study protein structures and functions
• May be studied in vitro, in vivo, and in silico.
• In vitro (Latin: in glass) are performed with cells or biological
molecules studied outside their normal biological context; eg.
proteins are examined in solution, cells in artificial culture medium.
Known as "test tube experiments", these studies in biology are
traditionally done in test-tubes first before proceeding to in vivo
studies.
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4. Protein methods
• In vivo (Latin for "within the living”) experiments can provide
information about the physiological role of a protein in the context
of a cell or even a whole organism. The effects of various biological
entities are tested on whole, living organisms as opposed to those
done in vitro. Examples – animal drug testing
• In silico studies use computational methods to study proteins.
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5. Protein purification : Analytical and
preparative methods
• Can roughly be divided into analytical and preparative methods.
• Depends on the amount of protein that can be purified with that
method.
• Analytical methods - aim to detect and identify a protein in a
mixture, usually prepared in a small amount, eg protein function
and structure studies.
• Preparative methods - aim to produce large quantities of the
protein for other purposes, such as industrial use,
biopharmaceutical products.
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6. The purification of proteins is an essential
first step in understanding their function
• For detecting proteins, for isolating and purifying proteins, and
for characterizing the structure and function of proteins, often
requiring that the protein first be purified.
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Never waste pure
thoughts on an
impure protein!
Sequence
Function
Structure

7. The assay: How do you recognize the
protein that you are looking for?
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You need an biological assay!
You need to know what the protein does. For example,
Substrate A  Product B
Protein C
If the protein C converts substrate A to be Product B under
some specific condition, then this reaction is your assay.
If Product B is formed, your protein is present and active
If Product B is not formed , your protein is absent, or may be
present, but is inactive

9. Proteins must be released from cells to be
purified
• Protein purification - multiple steps processes to isolate one or a
few proteins from a complex mixture, usually cells, tissues or
whole organisms.
• The purification process may separate the protein and non-protein
parts of the mixture, and finally separate the desired protein from
all other proteins.
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Proteins can be purified
based on differences in
solubility, size, charge
and binding affinity.

10. Protein purification
• Preliminary steps
– Extraction
– Differential centrifugation
– Salting out
• Purification steps
– Size exclusion chromatography
– Separation based on charge or hydrophobicity
– Affinity chromatography: metal binding, immunoaffinity chromatography,
tagged protein purification
– HPLC
• Concentration of the purified protein
– Lyophilization
– Ultrafiltration
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11. Protein extraction
• Mostly performed at 4°C to minimize protein degradation and
denaturation, to minimize the loss of biological activity
• Homogenization : Break open the cells and subcellular organelles,
such as mitochondria, nucleus and endoplasmic reticulum to
release the protein
• Homogenization methods:
– Sonication
– Lysis by high pressure, filtration
– High force shearing
– Permeabilization by organic solvents or enzymatic treatment
• The method of choice depends on how fragile the protein is and
how sturdy the cells are eg, bacterial cell, plant cell.
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13. Protein extraction buffer
• After this extraction process soluble proteins will be suspended in the
protein extraction buffer.
• The choice of the buffer system, depends on:
– the stability of the target protein with respect to pH and the
buffering compound.
– the purification procedure. To avoid time and protein loss caused by
an additional buffer exchange step, it is advisable to choose a buffer
that is compatible with the first chromatography step.
• Buffer, depends on the pH range
– HEPES: pH 7.2 – 8.2
– Tris.HCl: pH 7.0 - 9.0
– Sodium dihydrogen phosphate: pH 5.8 – 8.0
• Protease inhibitor – PMSF, leupeptin (to inhibit proteases)
• Detergent – SDS, Triton X, Tween (to solubilize proteins) 13

14. Protein extraction buffer ingredient
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15. Differential centrifugation
• After the cells are homogenized, the proteins can be
separated from cell membranes, DNA etc. by differential
centrifugation.
• Centrifugation is a process that involves the use of the
centrifugal force for the separation of mixtures.
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YOU MUST BALANCE
THE SAMPLES
BEFORE
CENTRIFUGATION!!!

16. Differential centrifugation
• You do this by increasing the effective gravitational force
on a tube so as to more rapidly and completely cause the
precipitate ("pellet") to gather on the bottom of the tube.
• The solution/supernatant is then either quickly decanted
from the tube without disturbing the precipitate or
withdrawn with a pipette tip.
• The rate of centrifugation is specified by the acceleration
applied to the sample, typically measured in revolutions
per minute (RPM) or g.
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17. Differential centrifugation
Cells are disrupted in a
homogenizer and the
resulting mixture, called
homogenate, is centrifuged in
a step-wise manner with
increasing centrifugal force.
The denser material will form
a pellet at lower centrifugal
force than the less-dense
material.
The isolated fractions can be
used for further purification.

18. Differential centrifugation
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Supernatant
Separation is based on size and density,
with larger and denser particles
pelleting at lower centrifugal forces.
Smaller cell fragments and organelles
remain in the supernatant and require
more force and greater times to pellet.
Increasing speed

19. Salting out
• After the protein are solubilized, they are subjected to a crude
purification based on solubility.
• Most proteins are less soluble at high salt concentration, an
effect known as “salting out”
• Ammonium sulfate is commonly used, the salt concentration
at which a protein precipitates differs from one protein to
another
• Salting out can be used to fractionate proteins.
• Salting out can be used to concentrate dilute solutions of
proteins.
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20. Salting out
• Solubility of different proteins varies at different salt
concentration
• You will end up with different fractions of proteins that were
precipitated at: 0.2, 0.4, 0.6, 0.8 and 1.0 M
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0.2 M
fraction
0.4 M
fraction
0.8 M
fraction
0.6 M
fraction
1.0 M
fraction

21. Dialysis
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• Ammonium sulfate salt can be
removed from protein by dialysis.
• Salt smaller than the pore size of
the membrane pass through the
dialysis bag.
• Protein larger than the pore size
will be retained.
• You normally dialyse overnight ,
in buffer 200-500X the volume of your
sample with buffer changed every few
hours

22. Chromatography
• Consists of a variety of biomolecule separation techniques
• Biomolecules mixtures is dissolved in mobile phase, which
carries it through a structure holding another material called
stationary phase.
• Various biomolecules are separated based on their different
retention times on the stationary phase.
• Biomolecules that spend more time in the stationary phase will
move slower.
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Based on this
difference,
biomolecules will
be separated.

23. Types of chromatography
• Based on type of support
– Column
– Planar : Paper and Thin Layer chromatography (TLC)
• Based on retarding forces
– Gel filtration/size-exclusion chromatography
– Ion exchange
– Affinity chromatography
• Based on mobile phase
– Gas chromatography: Gas-liquid, Gas-solid
– Liquid chromatography
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24. Column chromatography
• The material that makes up the
stationary phase is packed in a
column.
• The sample is a small volume of
concentrated solution that is
applied to the top of the column.
• The mobile phase, called that
eluent is passed through the
column.
• As the eluent flows through the
column, the compounds of the
sample migrate at different rates.
• Eluate is collected and analyzed
for the presence of a particular
protein.
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25. Planar Chromatography
• Support is paper or TLC plates.
• Sample do not elute out but
form spots on the plate/paper.
• To identify, calculate retention
factor (Rf) and compare to Rf
values of known compounds.
• Rf is the ratio of distance
traveled by samples divided by
distance traveled by solvent
front.
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26. Size exclusion/Gel filtration chromatography
• Separation based on protein size
• Column filled with insoluble, but highly
hydrated beads.
• Beads have pores, made of dextran,
agarose or polyacrylamide (Sephadex,
Sepharose, Biogel)
• Molecules smaller than the pore size of
the beads can move in and out of the
gel, travel a longer path, will take
longer to move through the column
• Molecules larger than the pore size
move in between the beads, move
along faster
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Larger proteins elute first,
smaller protein elute later.

27. Ion exchange chromatography
Separation based on the protein net charge
• If the column has positively charged
stationary phase, negatively charged
molecules are bound and positively charged
molecules are in the unbound fraction and
washed out earlier
• Later, the negatively charged molecules can
be eluted out by releasing it from the
column.
• This can be done by adding a negatively
charged salt that can compete for the
positively charged stationary phase.
• The salt will replace the bound proteins,
releasing the bound proteins.
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28. Ion exchange chromatography
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For separation of positively charge molecule, a cation
exchanger bead is used instead.

29. Affinity chromatography
• Affinity chromatography is based
on selective non-covalent
interaction between antigen and
antibody, enzyme and substrate,
or receptor and ligand.
• These fusion proteins are labelled
with specific tags such as His-tags,
biotin or antigens, which bind to
the stationary phase specifically.
• After purification, some of these
tags may be removed and the
pure protein is obtained.
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30. High performance liquid chromatography
(HPLC)
• A form of chromatography applying high pressure to drive the
solutes through the column faster.
• This means that the diffusion is limited and the resolution is
improved.
• The most common form is "reversed phase" HPLC, where the
column material is hydrophobic.
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31. High performance liquid chromatography
(HPLC)
• The proteins are eluted by a gradient of increasing amounts
of an organic solvent, such as acetonitrile.
• The proteins elute according to their hydrophobicity.
• After purification by HPLC the protein is in a solution that
only contains volatile compounds, and can easily be
lyophilized.
• HPLC purification frequently results in denaturation of the
purified proteins and is thus not applicable to proteins that
do not spontaneously refold.
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32. High performance liquid chromatography
(HPLC)
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33. High performance liquid chromatography
(HPLC)
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34. Chromatogram
• The HPLC result printout of the
peaks that is produced is
called a chromatogram.
From a chromatogram, you can
1. Identify the compound
2. Determine the concentration
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35. Study questions
1. Why is it not advisable to work with impure proteins?
2. Why is it important to perform most of the protein purification
procedure at 4°C?
3. What do you expect to find in the protein mixture after
homogenization?
4. How does differential centrifugation separate protein mixtures?
5. How do proteins fractionated by salting out?
6. Name the types of chromatography used for protein purification.
7. What are the differences between gel filtration, ion exchange and
affinity chromatography?
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