PCB 4233L QUIZ 2: LABS 4-6 VERIFIED ACCURATE
STUDY GUIDE
UNIT 4: ANION EXCHANGE CHROMATOGRAPHY EXPERIMENT - Answers - Goal:
- separate charged proteins
- obtain purified protein [IgG (gamma immunoglobulin) and negatively charged protein]
- negatively charged proteins and contaminants (also negatively charged) will bind to
the column
- Gamma immunoglobulins are positively charged at pH 8 and do not bind to the
column. We expect the IgGs to flow through the column and be found in the initial flow
through
- the column is washed with additional buffer to ensure all neutral and positively charged
proteins are removed
- the negatively charged proteins are captured in the column and eluted with a high salt
buffer. The chloride ions compete with proteins for access to the positively charged
matrix, displacing the negatively charged proteins and allowing them to be collected in
the flow-through.
Chromatography - Answers - A technique that is used to separate the components of a
mixture by differential movement through a two-phase system, with one phase being a
liquid and the other an insoluble matrix.
Column chromatography - Answers - a form of partition, adsorption, ion exchange, or
affinity chromatography in which one phase is liquid flowing down a column packed with
the second phase, a solid; the dissolved substances form a partition between teh solid
and liquid phases depending on the chemical and physical conditions of each phase
Ion exchange chromatography - Answers - the chromatographic procedure in which
cationic or anionic proteins in the mobile (liquid) phase are separated by electrostatic
interactions with the stationary (solid) phase
Cation exchange - Answers - the process in which a cation (+ charge) in a liquid phase
exchanges with another cation present as the counter ion of a negatively charged solid
polymer (cation exchanger).
a cation exchanger is an insoluble solid that has negatively charged radicals attached to
it that can attract and hold cations that pass by within a moving solution if they are more
attracted to the acid groups than the counter ion present (such as chlorine, carbonate,
or phosphate ions).
Carboxymethylcellulose (CM) is a common cation exchanger used to separate
positively charged proteins
, Anion exchange - Answers - the process in which an anion (- charge) in a liquid phase
exchanges with another anion previously bound to a solid, positively charged phase, the
latter being an anion exchanger.
an anion exchanger is an insoluble solid with cation groups that can attract and hold
anions that pass by in a moving solution in exchange for anions previously held.
Diethylaminoethyl (DEAE) cellulose is a common anion exchanger used to separate
negatively charged proteins.
1. Anion Exchange Chromatography - Answers - Purify your salt-precipitated protein by
anion exchange
Anion exchange chromatography - Answers - Performed using ammonium sulfate
precipitated fraction of goat serum, which was later dialyzed.
The anion exchange spin column uses membrane-adsorbent technology as a
chromatographic matrix to fractionate charged proteins.
The Pierce Strong Ion Exchange Spin Column Q is a strong anion exchanger. The
membrane is positively charged and exchanges with negatively charged particles.
In our case, when the pH is 8, our protein will have a net positive charge when the
charge from each amino acid is summed.
The spin column Q - Answers - Has a binding capacity of 4 mg of protein per 0.4 mL of
volume, but needs to be equilibrated before a sample is added.
The column is equilibrated by passing a low salt buffer through it.
This ensures that the membrane is ready to adsorb negatively charged particles.
Procedure: spin column anion exchange chromatography - Answers - - The sample will
be added to the column and spun.
- Once it is applied, those proteins that are negatively charged will bind to the positively
charged resin beads.
- Gamma immunoglobulins are slightly positively charged at pH 8 and thus do not bind
to the column.
- Contaminating proteins, which have a negative charge, will bind to the column.
- We expect our immunoglobulins to flow through the column and they will be found in
the initial flow through.
- The column is then washed with additional buffer to ensure that all neutral and
positively charged proteins that have no affinity towards the matrix are removed.
- The negatively charged particles elute from the column when the high salt buffer is
added to the column.
- The chloride ions begin to compete with the proteins for access to the positively
charged matrix as the concentration of salt increases, displacing the negatively charged
proteins and allowing them to be collected in the flow-through.
Purification conditions - Answers - Sample: Ammonium sulfate precipitated fraction of
serum IgGs that was desalted by dialysis
Buffer A: 25 mM Tris*HCl buffer, pH 8.0
STUDY GUIDE
UNIT 4: ANION EXCHANGE CHROMATOGRAPHY EXPERIMENT - Answers - Goal:
- separate charged proteins
- obtain purified protein [IgG (gamma immunoglobulin) and negatively charged protein]
- negatively charged proteins and contaminants (also negatively charged) will bind to
the column
- Gamma immunoglobulins are positively charged at pH 8 and do not bind to the
column. We expect the IgGs to flow through the column and be found in the initial flow
through
- the column is washed with additional buffer to ensure all neutral and positively charged
proteins are removed
- the negatively charged proteins are captured in the column and eluted with a high salt
buffer. The chloride ions compete with proteins for access to the positively charged
matrix, displacing the negatively charged proteins and allowing them to be collected in
the flow-through.
Chromatography - Answers - A technique that is used to separate the components of a
mixture by differential movement through a two-phase system, with one phase being a
liquid and the other an insoluble matrix.
Column chromatography - Answers - a form of partition, adsorption, ion exchange, or
affinity chromatography in which one phase is liquid flowing down a column packed with
the second phase, a solid; the dissolved substances form a partition between teh solid
and liquid phases depending on the chemical and physical conditions of each phase
Ion exchange chromatography - Answers - the chromatographic procedure in which
cationic or anionic proteins in the mobile (liquid) phase are separated by electrostatic
interactions with the stationary (solid) phase
Cation exchange - Answers - the process in which a cation (+ charge) in a liquid phase
exchanges with another cation present as the counter ion of a negatively charged solid
polymer (cation exchanger).
a cation exchanger is an insoluble solid that has negatively charged radicals attached to
it that can attract and hold cations that pass by within a moving solution if they are more
attracted to the acid groups than the counter ion present (such as chlorine, carbonate,
or phosphate ions).
Carboxymethylcellulose (CM) is a common cation exchanger used to separate
positively charged proteins
, Anion exchange - Answers - the process in which an anion (- charge) in a liquid phase
exchanges with another anion previously bound to a solid, positively charged phase, the
latter being an anion exchanger.
an anion exchanger is an insoluble solid with cation groups that can attract and hold
anions that pass by in a moving solution in exchange for anions previously held.
Diethylaminoethyl (DEAE) cellulose is a common anion exchanger used to separate
negatively charged proteins.
1. Anion Exchange Chromatography - Answers - Purify your salt-precipitated protein by
anion exchange
Anion exchange chromatography - Answers - Performed using ammonium sulfate
precipitated fraction of goat serum, which was later dialyzed.
The anion exchange spin column uses membrane-adsorbent technology as a
chromatographic matrix to fractionate charged proteins.
The Pierce Strong Ion Exchange Spin Column Q is a strong anion exchanger. The
membrane is positively charged and exchanges with negatively charged particles.
In our case, when the pH is 8, our protein will have a net positive charge when the
charge from each amino acid is summed.
The spin column Q - Answers - Has a binding capacity of 4 mg of protein per 0.4 mL of
volume, but needs to be equilibrated before a sample is added.
The column is equilibrated by passing a low salt buffer through it.
This ensures that the membrane is ready to adsorb negatively charged particles.
Procedure: spin column anion exchange chromatography - Answers - - The sample will
be added to the column and spun.
- Once it is applied, those proteins that are negatively charged will bind to the positively
charged resin beads.
- Gamma immunoglobulins are slightly positively charged at pH 8 and thus do not bind
to the column.
- Contaminating proteins, which have a negative charge, will bind to the column.
- We expect our immunoglobulins to flow through the column and they will be found in
the initial flow through.
- The column is then washed with additional buffer to ensure that all neutral and
positively charged proteins that have no affinity towards the matrix are removed.
- The negatively charged particles elute from the column when the high salt buffer is
added to the column.
- The chloride ions begin to compete with the proteins for access to the positively
charged matrix as the concentration of salt increases, displacing the negatively charged
proteins and allowing them to be collected in the flow-through.
Purification conditions - Answers - Sample: Ammonium sulfate precipitated fraction of
serum IgGs that was desalted by dialysis
Buffer A: 25 mM Tris*HCl buffer, pH 8.0
