BY1101 Introduction to Molecular and Cellular Biology
Tutorial for module BY1101:
Chromatography
Joe Colgan (tcolgan@tcd.ie)
BY1101 Introduction to Molecular and Cellular Biology
Tutorial objectives
• Describe chromatography
• Describe the different types and why they
were used in the BY1101 practicals
BY1101 Introduction to Molecular and Cellular Biology
Column chromatography
• What is chromatography?
Column
chromatography
• A set of lab techniques to separate mixtures
• Mobile phase:
• Fluid that houses the mixture to be separated
• (e.g. Cell lysate, haemoglobin, mould extract)
• Stationary phase:
• Structure holding another material that interacts with
and aids in separation of mixture
• (e.g. Sepharose, sephadex, DEAE-cellulose)
Mobile
Stationary
BY1101 Introduction to Molecular and Cellular Biology
Three types of column chromatography used in the
BY1101 practicals. What are they?
Practical 2
Practical 3
Experiment 1
Practical 3
Experiment 2
Affinity
chromatography
Gel filtration
chromatography
Ion exchange
chromatography
Separates molecules
based on biological
specificity
Separate molecules
based on size
Separates molecules
based on charge
BY1101 Introduction to Molecular and Cellular Biology
Affinity chromatography
What do we use affinity chromatography for?
Purpose
•Purification and concentration of biomolecules,
such as proteins, on the basis of their biological
specificity
Applications
•Purification of antibodies
•Purification of enzymes
BY1101 Introduction to Molecular and Cellular Biology
Affinity chromatography
What was the purpose of Practical 2 – Experiment One?
Glutathione S-transferase (GST):
• Catalyzes conjugations of the substrate glutathione (GSH)
•GST is an enzyme and binds GSH in an enzyme-substrate
complex
Enzyme (E) + Substrate (S)  Enzyme-substrate complex
•Interaction is dynamic and GST will bind reversibly
Substrate
Enzyme
GSH
GST
Enzyme-substrate
complex
BY1101 Introduction to Molecular and Cellular Biology
Affinity chromatography
What is the source of
the GST?
Natural source
Recombinant protein
BY1101 Introduction to Molecular and Cellular Biology
Affinity chromatography
What is a recombinant protein?
A protein encoded for by a gene – recombinant DNA – that has been cloned into a
system that supports its transcription and translation
l
P
O
gst gene
Transcription
mRNA
Translation
l= lac operon
P= Promoter
O= Operator
Repressor
protein
BY1101 Introduction to Molecular and Cellular Biology
Affinity chromatography
How do we get “overexpression” of a protein?
Isopropyl-beta-D-thio-galactoside (IPTG):
IPTG binds to the repressor protein and inactivates it
l
P
O
gst gene
mRNA
Transcription of mRNA
Repressor
protein
IPTG
l= lac operon
P= Promoter
O= Operator
Within the present experiment, what would be the purpose of adding IPTG to
the bacterial culture?
BY1101 Introduction to Molecular and Cellular Biology
Molecule of interest
Mobile phase
Stationary phase
Escherichia coli
(bacterial) lysate
Sepharose beads coated
with glutathione
Glutathione S-transferase
Are the proteins present in the cell lysate in their native (active) state or are
they denatured?
Would you expect the proteins present in the lysate to exhibit their natural
biological activity?
Why was it important to keep the cell lysate on ice?
BY1101 Introduction to Molecular and Cellular Biology
Affinity chromatography
Sepharose
Glutathione (GSH)
Cell lysate
Glutathione Stransferase (GST)
Sepharose-Glutathione
Stationary phase
Mobile phase
BY1101 Introduction to Molecular and Cellular Biology
Mobile
Stationary
1. Pour the column
(Sepharose Beads-Stationary phase)
2. Wash the column
(Phosphate buffered saline)
BY1101 Introduction to Molecular and Cellular Biology
3. Run the column
(E. coli lysate) Contains enzyme of interest
- GST within lysate binds to glutathionesepharose beads complex
Would you expect GST to be in fraction
one?
Mobile
Stationary
4.(PBS) Washes the column
- Other bacterial proteins are washed out of
the column leaving only GST bound to
sepharose beads
Why does GST remain in the column
after the PBS washes?
1
2
3
4
5
6
BY1101 Introduction to Molecular and Cellular Biology
3. Run the column
(E. coli lysate) Contains enzyme of interest
- GST within lysate binds to glutathionesepharose beads complex
4. (PBS) Washes the column
- Other bacterial proteins are washed out of
the column leaving only GST bound to
sepharose beads
Mobile
Stationary
5. (Glutathione) Substrate of enzyme
- High concentration of glutathione
displaces GST from the beads, binds to GST
and is eluted out of the column
1
2
3
4
5
6
7
BY1101 Introduction to Molecular and Cellular Biology
Affinity chromatography
GST binds to GSHSepharose beads
Free GSH bind GST
and elutes
Non-bound proteins
removed
Column is
washed
All proteins in
lysate except GST
Addition of
free GSH
1
Purified
GST
7
BY1101 Introduction to Molecular and Cellular Biology
SDS-PAGE preparation
• Precipitation of protein with trichloroacetic acid (TCA)
• Pellet the precipitated protein by centrifugation
• Dissolve precipitated protein in sodium-dodecyl sulphate (SDS)
• Boil the protein samples
BY1101 Introduction to Molecular and Cellular Biology
Gel filtration chromatography
Practical 3- Experiment One
Purpose
Separation of macromolecules based on size
Applications
•Determination of relative molecular size
•Separation of molecules on the basis of size
•Removal of inorganic ions from preparation of
protein
BY1101 Introduction to Molecular and Cellular Biology
Gel filtration chromatography
Direction of flow
BY1101 Introduction to Molecular and Cellular Biology
Gel filtration chromatography
Practical 3- Molecule of interest
Haemoglobin
Purple/red colour
Oxygen-depleted blood
Venous blood
Oxyhaemoglobin
Scarlet/red colour
Oxygen-rich blood
Arterial blood
BY1101 Introduction to Molecular and Cellular Biology
Gel filtration chromatography
Practical 3- Molecule of interest
Haemoglobin
Methaemoglobin
Oxidation
Haemoglobin
Reduction
Oxidation: Potassium ferricyanide + Haemoglobin  Oxidised haemoglobin (methaemoglobin)
Reduction: Ferrous sulphate + methaemoglobin  Reduced haemoglobin
BY1101 Introduction to Molecular and Cellular Biology
Mobile
Stationary
1. Pour the column
(G-25 Sephadex beads-Stationary phase)
2. Wash the column
(20mM PBS, pH 7)
BY1101 Introduction to Molecular and Cellular Biology
3. Add the reducing agent
(40mM FESO4 + 80mM Na2EDTA)
4. Add methaemoglobin
Mobile
Stationary
Methaemoglobin
Reducing agent
Haemoglobin
BY1101 Introduction to Molecular and Cellular Biology
Gel filtration chromatography
Addition of reducing
agent
Addition of
methaemoglobin
Reduction of
methaemoglobin to
haemoglobin
BY1101 Introduction to Molecular and Cellular Biology
Ion exchange chromatography
Purpose
Separation of molecules on the basis of charge
Applications
•Water softening, purification and
decontamination
BY1101 Introduction to Molecular and Cellular Biology
Ion exchange chromatography
Direction of flow
Separation of molecules on the basis of charge
BY1101 Introduction to Molecular and Cellular Biology
Ion exchange chromatography
Molecules of interest
Glucose oxidase
Catalase
Mobile phase
Stationary phase
Aspergillus niger
(fungal) extract
DEAE- Cellulose
BY1101 Introduction to Molecular and Cellular Biology
Ion exchange chromatography
• Stationary phase:
• Diethylaminoethyl (DEAE) cellulose
• Positively charged (protonated)
• Interacts with negatively charged molecules (anions)
• Anion exchanger: Stationary phase is positively charged
BY1101 Introduction to Molecular and Cellular Biology
Ion exchange chromatography
Separation of molecules on the basis of charge
Glucose oxidase:
•Oxidation of glucose to hydrogen peroxide and
glucono-1,5-lactone, which hydrolyzes to gluconic
acid
•Glucose oxidase (GO) requires cofactor flavin
adenine dinucleotide (FAD)
Glucose + GO:FAD  Glucono-1,5-lactone + GO:FADH2
Glucose oxidase
GO:FADH2 + O2  GO:FAD + H2O2
Substrate
Enzyme
Glucose
Glucose oxidase
Enzyme-substrate
complex
BY1101 Introduction to Molecular and Cellular Biology
Ion exchange chromatography
Separation of molecules on the basis of charge
Catalase:
•Catalyzes the decomposition of hydrogen peroxide
(H2O2) into water (H2O) and oxygen (O2)
2H2O2  2H2O + O2
Catalase
Substrate
Enzyme
Hydrogen peroxide
Catalase
Enzyme-substrate
complex
BY1101 Introduction to Molecular and Cellular Biology
Mobile
Stationary
1. Pour the column
(DEAE-Cellulose-Stationary phase)
2. Wash the column
(PBS)
BY1101 Introduction to Molecular and Cellular Biology
Mobile
Stationary
3. Run the column
(Mould extract-Aspergillus niger)
4. Wash the column
(Buffer 1- 20mM NaOAc, 5mM acetic acid)
How do we get our enzymes of interest out of
the column?
BY1101 Introduction to Molecular and Cellular Biology
3. Run the column
(Mould extract-Aspergillus niger)
4. Release the bound molecules
(Buffer 1- 20mM NaOAc, 5mM acetic acid)
(Buffer 2- 40mM NaOAc, 40mM acetic acid)
(Buffer 3-100mM NaOAc, 100mM acetic acid)
Mobile
Stationary
•Lowering pH neutralizes negative charge on the
protein molecules
•Increase anionic molecules for competition
1
2
3
4
5
6
7
BY1101 Introduction to Molecular and Cellular Biology
Ion exchange chromatography
Buffer I
20mM sodium acetate
5mM acetic acid
Buffer II
Buffer III
Buffer II elutes catalase
Buffer III elutes glucose oxidase
40mM sodium acetate
100mM sodium acetate
40mM acetic acid
100mM acetic acid
Catalase
Glucose oxidase
BY1101 Introduction to Molecular and Cellular Biology
Ion exchange chromatography
Add molecules of
varying ionic charge
Negatively charged
molecules bind to beads
Addition of different
buffers changes charge
BY1101 Introduction to Molecular and Cellular Biology
Functional assays
In biological studies, what is the role of control samples?
Catalase
Control
Glucose
Glucose oxidase
Hydrogen
peroxide
(H2O2)
Control
Glucose
Hydrogen
peroxide
(H2O2)
BY1101 Introduction to Molecular and Cellular Biology
Summary
Chromatography: Used to separate out
mixtures
Affinity
chromatography
Gel filtration
chromatography
Ion exchange
chromatography
Separates molecules
based on biological
specificity
Separate molecules
based on size
Separates molecules
based on charge
BY1101 Introduction to Molecular and Cellular Biology
MCQ Advice
• Get your lab books up to date (e.g. Tables, graphs)
• If you have problems with the questions ask a
demonstrator (or me)
• When it comes to the exam, revise all of the lab book
(including the introductory notes)
• Read over lab slides available on the teaching website
BY1101 Introduction to Molecular and Cellular Biology
Next week
- Developmental biology: Embryology
- Lectures 3, 4 and 5
- Campbell: Chapter 47 (section 47.1)