Worksheet: EXPERIMENT 2.3 Purifying
proteins by ion exchange chromatography
02 November 2020
11:47
INTRODUCTION
The study of protein structure and function is generally more straightforward if highly
purified samples are available. This allows the concentration of the protein under
study to be determined accurately (important for quantification of catalytic activity for
example), is vital for structure determination and to ascribe an observed function to
that protein. Most proteins, however, need to be isolated from the thousands of
other proteins that are present in eukaryotic or prokaryotic cells that are used to
over-express them. A typical protein purification workflow typically involves cell
disruption, fractionation (typically using centrifugation) followed by chromatography.
Chromatography exploits differences in the chemical and physical properties of
proteins such as the net charge on a protein at a defined pH (ion-exchange (this
practical)) and its size (size exclusion chromatography). As many proteins may have
the same size or pI it is often necessary to run two or more chromatographic
techniques each fractionating using a different characteristic (known as orthogonal
methods) as few proteins have the same pI AND mass. Here we will investigate how
a simple binary mixture of two, coloured proteins can be separated using ion-
exchange chromatography.
PREPARATION
Before you come to the session, you should:
· View the background materials on Minerva (Introduction to Experiment 2.3) and
relevant lecture material (Project 2: Separating Biological Molecules)
· Read through the session worksheet so you understand what you will be doing
(but don’t start to complete it).
· Assess your understanding of this method by:
(i) attempting the MRQs on Minerva (Preparatory quiz for Experiment 2.3).
(ii) Answering questions 1- 4 below.
1. CM- and DEAE-cellulose are examples of ion exchange chromatography (IEX)
resins. What are their full names, and by looking at their structures, deduce which is
a cation exchanger and which is an anion exchanger.
, CM-Cellulose full name is: carboxymethyl cellulose and it is negatively charged, so it
binds to positively charged proteins , so it is a cation exchanger.
STRUCTURE:
DEAE-cellulose's full name is: diethylaminoethyl cellulose and it is a positively
charged resin that binds to negatively charged proteins so it is an anion exchanger.
STRUCTURE:
proteins by ion exchange chromatography
02 November 2020
11:47
INTRODUCTION
The study of protein structure and function is generally more straightforward if highly
purified samples are available. This allows the concentration of the protein under
study to be determined accurately (important for quantification of catalytic activity for
example), is vital for structure determination and to ascribe an observed function to
that protein. Most proteins, however, need to be isolated from the thousands of
other proteins that are present in eukaryotic or prokaryotic cells that are used to
over-express them. A typical protein purification workflow typically involves cell
disruption, fractionation (typically using centrifugation) followed by chromatography.
Chromatography exploits differences in the chemical and physical properties of
proteins such as the net charge on a protein at a defined pH (ion-exchange (this
practical)) and its size (size exclusion chromatography). As many proteins may have
the same size or pI it is often necessary to run two or more chromatographic
techniques each fractionating using a different characteristic (known as orthogonal
methods) as few proteins have the same pI AND mass. Here we will investigate how
a simple binary mixture of two, coloured proteins can be separated using ion-
exchange chromatography.
PREPARATION
Before you come to the session, you should:
· View the background materials on Minerva (Introduction to Experiment 2.3) and
relevant lecture material (Project 2: Separating Biological Molecules)
· Read through the session worksheet so you understand what you will be doing
(but don’t start to complete it).
· Assess your understanding of this method by:
(i) attempting the MRQs on Minerva (Preparatory quiz for Experiment 2.3).
(ii) Answering questions 1- 4 below.
1. CM- and DEAE-cellulose are examples of ion exchange chromatography (IEX)
resins. What are their full names, and by looking at their structures, deduce which is
a cation exchanger and which is an anion exchanger.
, CM-Cellulose full name is: carboxymethyl cellulose and it is negatively charged, so it
binds to positively charged proteins , so it is a cation exchanger.
STRUCTURE:
DEAE-cellulose's full name is: diethylaminoethyl cellulose and it is a positively
charged resin that binds to negatively charged proteins so it is an anion exchanger.
STRUCTURE:
