3. Affinity Chromatography Flashcards
What is the general principle?
Ligand immobilized on matrix. Biospecific interactions (which can be ionic, hydrophobic, or both) between ligand and target proteins. Unwanted proteins flow through.
What are the different types of general ligand-target combos?
Ab-antigen; ligand-receptor; substrate-enzyme.
List the steps
- Equilibration
- Load sample
- Wanted proteins bind, unwanted flow through.
- Wash with buffer to rid of excess unbound proteins.
- Elute bound proteins by changing buffer conditions (pH, ionic strength).
- Reequilibration.
Why do we equilibrate and reequilibrate?
Equilibration: ensures buffer condition is equivalent to that of protein conditions to prevent protein denaturation or precipitation.
Reequilibration: allows for the reuse of the column by washing with buffers containing denaturants and chaotropes to remove residual bound proteins, preventing contamination.
What are the characteristics of the matrix and give examples.
- Physically withstand high pressure.
- Chemically withstand harsh conditions like pH changes.
Eg. Sepharose: cross-linked agarose that resists denaturation.
What are the characteristics of the ligand and give examples.
- Highly specific to target protein and easily coupled to matrix.
- Able to form stable and reversible complexes with target proteins.
- Able to dissociate easily from proteins without any adverse biochemical changes.
Eg. Small molecules like glutathione; macromolecule like protein A (group specific), lectin, Abs (monospecific)
What is a common issue in AC?
Steric hindrance
Explain the common issue in AC.
It is when proteins are not able to bind optimally to the ligand due to their size or conformation.
Explain the solution to the common issue in AC.
Use spacer arms which extend the ligand away from the matrix, allowing proteins to bind optimally to the ligand.
What is another issue with the solution to the common problem in AC and how do we fix it?
Spacer arms consist of inert hydrophobic chain structures that can form non-specific hydrophobic interactions. So we have to reduce the length of the spacer arms as much as possible.
How do we couple the ligand to the matrix? Give an example and explain.
Via derivatisation. Like cyanogen bromide activation. Hydroxyl groups are activated by CNBr and add to surface of matrix where they form cyanate esters, allowing for amino-group containing ligands to covalent bind with the matrix.
What are the 4 ways to elute proteins?
- Change buffer pH
- Change buffer ionic strength
- Use denaturants
- Use competitors (eg. Free reduced glutathione displaces GST-bound fusion proteins)
List the 3 elution methods, their conditions, and principles.
- Step elution, stepwise change: conditions of buffer change at the end of the process where column is free of contaminants and only bound proteins are left.
- Gradient elution, linear change: conditions of buffer change gradually throughout the process where weakly bound proteins elute first.
- Isocratic elution, no change in conditions.
Compare the peaks of gradient and isocratic elutions.
Gradient results in narrower peaks which indicate better separation and resolution. Also results in a more complete elution hence more frequent peaks.
Isocratic has wider peaks. Only target proteins get detected.
What are the 3 ligand-target combos?
- Streptavidin-biotin
- Protein A/G-Ig
- Lectin-glycan
Explain streptavidin-biotin.
Proteins get biotinylated and then loaded. Biotin region of proteins interact with streptavidin which is the ligand.
Explain protein A/G-Ig.
Proteins become fusion proteins with Ig binding domains which interacts with protein A/G which is the ligand.
Where do protein A and G come from?
A comes from S. aureus. G comes from Streptococcal bacteria.
What human and mouse subclasses do proteins A/G bind to?
All human Ig subclasses and all mouse IgG subclasses except for IgA, IgM, and serum albumin.
What are lectins?
Plant-derived macromolecules that bind to sugars.
What are lectin-glycans used for?
Purifying glycoproteins and differentiating terminal sugars in glycans.
State the different types of lectins, their source, what they bind to, and the eluting sugar.
- Concanavalin A; jackbean seeds; a-D-mannose and a-D-glucose; a-D-methylmannose.
- Wheat germ agglutinin; wheat germs; N-acetyl-b-D-glucosamine and N-acetyl-b-D-neuraminic acid; N-acetyl-b-D-glucosamine.
- Pisum sativum lectin; peas; a-D-mannose; a-D-methylmannose.
- Soybean lectin; soybean; N-acetyl-b-D-galactosamine; N-acetyl-b-D-galactosamine.
How can fusion proteins be purified in affinity chromatography?
Epitope tags can be attached to proteins upstream or downstream to form fusion proteins where the ligand interacts with the tag and the tag can also be enzymatically cleaved to recover native protein.
What are the 5 types of tags?
- Hemagglutinin (HA) tag
- FLAG tag
- Glutathione-S-transferase (GST) tag
- 6xHis tag
- Maltose-binding protein (MBP) tag
