Lecture 9 Flashcards
What is the properties of Ion-exchange chromatography
Separates on basis of net charge
• Column matrix (stationary phase):
• Cation exchangers: resin has
bound anionic groups (neg charge), e.g. carboxymethylcellulose
• Anion exchangers: resin has bound cationic groups (pos charge), e.g. DEAE‐cellulose
• Buffer at specific pH (mobile phase)
How does ion-exchange chromatography separate protein
Separation on basis of net charge
• Affinity of each protein for the charged groups on the column is affected by
• Magnitude of charge is
important (i.e. large net charge opposite to resin will elute at later time)
particles with the strongest opposite charge will be discharge the last
Anion exchange: bound anion (i.e. anion exchange will selectively purify negatively charged proteins
DEAE side chain [diethyl amino ethyl]
Cation exchange: bound cation (i.e. cation exchange will selectively purify positively charged proteins) CM side chain [carboxy methyl]
How is the elution of bound protein be achieved in ion-exchange chromatography
- change in pH (remove net charge from the protein)
* change in salt concentration
What is the properties of affinity chromatography
Separates on basis of binding affinity
•Column matrix (stationary phase): covalently linked ligand
•Protein with affinity for ligand > migration will be retarded
•Protein with no affinity for ligand > washed out of column in mobile phase
•Bound protein eluted by solution containing:
•High [salt]
•High [ligand]
Exp: conA:agglutinates RBC
:binds glucose/mannose in glycoproteins
What is an example of affinity chromatography
Separation of serine proteases (e.g. Thrombin or Factor Xa) from
cell free extract using Sepharose beads conjugated to
benzamidine
• Affinity of Thrombin for Benzamidine
• Stationary phase: Sepharose linked Benzamidine
• Mobile phase: Buffer to promote Thrombin‐benzamidine interaction
• Elution buffer: Competitive ligand or pH change
Successful affinity chromatography requires
- Biospecific ligand covalently attached to chromatography matrix
- Coupled ligand must retain specific binding affinity for target molecule
- Binding between ligand and target molecule must be reversible
How are antibodies used for purification?
- Bind directly to antigen at specific epitope on the target molecule
- Use with bacterial Immunoglobulin‐binding proteins (Protein‐A and Protein‐G)
‐ bind the constant region (Fc) of immunoglobulins
The use of ig binding proteins in purification
• Can be used to precipitate antigens directly from a
solution: immunoprecipitation
• Can be covalently linked
e.g. Agarose, Sepharose, Polyacrylamide, magnetic
beads,
Therefore, can use in column or batch purification
What are the example of general process in immunoprecipitation
- Mix protein sample (antigen) + soluble antibody (1)
- Incubate: protein‐antibody interaction (2)
- Add Protein‐A or Protein‐G: form insoluble antibody:protein complex (3)
- Centrifuge: pellet the antibody:protein complexes (4)
- Remove supernatant (unbound proteins)
- Elute: disrupt Ab:antigen (antibody:protein) interaction (5)
- Re‐centrifuge and collect supernatant (protein of interest)
What can you attach Ab to
(i) Sepharose or agarose beads
ii) Dynabeads (magnetic beads for biomagnetic separation
How to create a specific activity
- measured at each step of the purification process
- measure of enzyme purity
- measured in ‘units/mg’ of enzyme activity
Specific activity (units mg‐1) = units of activity (units)/amount of total protein (mg)
What is yield
Aim of purification: isolate protein of interest with maximum possible yield of intact protein
Yield (%) =
units of activity of given fraction/units of activity of starting material
Yield is measured at each step of purification process
What is FOLD PURIFICATION
In most cases, the specific activity of the pure material is not known
Purity is expressed relative to the starting material
Purification (fold) = Specific activity of a given fraction/Specific activity of starting material
