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

Stationar
y

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) Enzymesubstrate complex

Substrate
GSH

Interaction is dynamic and GST will bind

reversibly
Enzyme
Enzymesubstrate
complex
GST

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
Transcripti
on

gst gene

mRNA

Translation

Represso
r protein

l= lac
operon
P=
Promoter
O=
Operator

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

gst gene

mRNA
Transcription of
mRNA

l= lac
IPT
operon
Represso
G
P=
r protein
Promoter
Within the present experiment, what
would be the purpose of
O=
adding IPTG to the bacterial
culture?
Operator

BY1101 Introduction to Molecular and Cellular Biology

Molecule of interest

Mobile phase

Stationary phase

Escherichia coli
(bacterial) lysate

Sepharose beads
coated with
glutathione

Glutathione Stransferase

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
Sepharo
Glutathione
se

(GSH)

Cell
lysate

Glutathione Stransferase
(GST)

SepharoseGlutathione

Stationary phase

Mobile phase

BY1101 Introduction to Molecular and Cellular Biology

Mobile
Stationa
ry

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
glutathione-sepharose beads
Would
you expect GST to be
complex

Mobile

in fraction one?

Stationa
ry
4.(PBS) Washes the column
- Other bacterial proteins are
washed out of the column leaving
only GST
bound
sepharose
Why
does
GSTtoremain
in the
beads
column after the PBS

washes?
1

BY1101 Introduction to Molecular and Cellular Biology

3. Run the column

(E. coli lysate) Contains enzyme of
interest
- GST within lysate binds to
glutathione-sepharose beads
complex

Mobile
Stationa
ry

4. (PBS) Washes the column

- Other bacterial proteins are
washed out of the column leaving
only GST bound to sepharose
beads

5. (Glutathione) Substrate of enzyme

- High concentration of glutathione
displaces GST from the beads,
out
2 binds
3 to GST
4 and
5 is eluted
6
7 of
the column

BY1101 Introduction to Molecular and Cellular Biology

Affinity chromatography
GST binds to
GSH-Sepharose
beads

Free GSH bind

GST and elutes

Non-bound
proteins
removed
Column
is
washed

All proteins in
lysate except
GST

Addition
of free
GSH

Purifie
dGST

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
Oxidatio
n

Haemoglobin
Reducti
on

Oxidation: Potassium ferricyanide + Haemoglobin Oxidised haemoglobin

(methaemoglobin)
Reduction: Ferrous sulphate + methaemoglobin Reduced haemoglobin

BY1101 Introduction to Molecular and Cellular Biology

Mobile
Stationa
ry

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
Stationa
ry

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

Direction of fow

Ion exchange
chromatography
Separation of molecules on the basis of charge

BY1101 Introduction to Molecular and Cellular Biology

Molecules of
interest

Ion exchange
chromatography
Mobile phase
Stationary phase
Aspergillus niger
(fungal) extract

Glucose oxidase

Catalase

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 oxidase
Substrate
Glucose

Glucose + GO:FAD Glucono-1,5-lactone +

GO:FADH2
GO:FADH2 + O2 GO:FAD + H2O2
Enzyme
Enzymesubstrate
complex
Glucose oxidase

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)

Catalase

2H2O2 2H2O +
O2

Substrate

Enzyme

Hydrogen
peroxide

Catalase

Enzymesubstrate
complex

BY1101 Introduction to Molecular and Cellular Biology

Mobile
Stationa
ry

1. Pour the column

(DEAE-Cellulose-Stationary phase)
2. Wash the column
(PBS)

BY1101 Introduction to Molecular and Cellular Biology

Mobile
Stationa
ry

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
Lowering
pH neutralizes
3-100mM
NaOAc, negative
100mM
charge
on the protein molecules
acetic
acid)
Increase anionic molecules for
competition

Mobile
Stationa
ry

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

Catalase

100mM acetic acid

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

Hydroge
n
peroxide
(H2O2)

Control

Glucose

Hydroge
n
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)