What is Affinity chromatography?
Affinity Chromatography is a method of separating and purifying biomolecules, such as proteins, nucleic acids, and other biomolecules from a complex mixture by using specific interactions between the biomolecule of interest and an immobilized ligand. The immobilized ligand is covalently attached to a solid support, such as beads or a column, and the sample containing the biomolecule of interest is passed through it. The biomolecule of interest will bind to the immobilized ligand while other molecules in the sample will not, allowing for separation and purification of the biomolecule of interest. Affinity Chromatography offers several advantages such as high specificity, selectivity, high binding capacity, and high purity of the purified biomolecule. It can be used for purifying a wide range of biomolecules but it has some limitations such as the need for a specific immobilized ligand, the need for high sample concentrations, the possibility of conformational changes in the purified biomolecule and the need for stringent washing conditions to remove non-specifically bound molecules.
Also Read: Affinity chromatography Principle, Types, Steps, Applications
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MCQ on Affinity chromatography
1. What is the principle of Affinity Chromatography?
a) Separation based on size
b) Separation based on charge
c) Separation based on specific binding interactions
d) Separation based on pH
2. What are some common types of Affinity Chromatography?
a) Protein A/G, His-tag, Glutathione, Nucleic acid
b) Size exclusion, Ion exchange, Reversed-phase
c) Gel filtration, centrifugation, ultracentrifugation
d) Gel electrophoresis, Western blot, ELISA
3. What are the advantages of Affinity Chromatography?
a) High specificity and selectivity
b) High binding capacity
c) High purity of the purified biomolecule
d) All of the above
4. What are the limitations of Affinity Chromatography?
a) The need for a specific immobilized ligand for the biomolecule of interest
b) The need for high sample concentrations
c) The possibility of conformational changes in the purified biomolecule
d) All of the above
5. How is the immobilized ligand in Affinity Chromatography prepared?
a) By covalently attaching it to a solid support
b) By adding it to the sample
c) By mixing it with the mobile phase
d) By heating the sample
6. How is the sample prepared for Affinity Chromatography?
a) In a buffer compatible with the immobilized ligand and the biomolecule of interest
b) In a high salt buffer
c) In a low pH buffer
d) In a high pH buffer
7. How is the purified biomolecule eluted from the immobilized ligand?
a) By changing the pH or temperature
b) By adding a competitive ligand
c) By using a high salt or chaotropic solution
d) All of the above
8. How can you determine if your purification is successful?
a) By analyzing the purity of the purified biomolecule
b) By running a western blot
c) By mass spectrometry
d) All of the above
9. Can Affinity Chromatography be used for downstream applications?
a) Yes
b) No
10. What buffer is used for binding in Affinity Chromatography?
a) Neutral or slightly alkaline buffer
b) Low salt buffer
c) High salt buffer
d) High pH buffer
11. What is the most common solid support used in Affinity Chromatography?
a) Agarose beads
b) Sepharose beads
c) Cellulose beads
d) Silica beads
12. What is the most common immobilized ligand used in Affinity Chromatography?
a) Protein A or G
b) Histidine tag
c) Glutathione S-transferase tag
d) Biotin
13. What reagents are used to detect and quantify the purified biomolecule in Affinity Chromatography?
a) Stains or dyes
b) Antibodies
c) mass spectrometry
d) All of the above
14. What are some downstream applications of Affinity Chromatography?
a) Protein crystallography
b) Enzymatic assays
c) Structural biology
d) All of the above
15. In which order should the following steps be performed in Affinity Chromatography: Sample preparation, Binding, Washing and Elution?
a) Sample preparation, Elution, Washing, Binding
b) Binding,
c) Sample preparation, Binding, Washing, Elution
d) Washing, Binding, Elution, Sample preparation
Answer Key
- c) Separation based on specific binding interactions
- a) Protein A/G, His-tag, Glutathione, Nucleic acid
- d) All of the above
- d) All of the above
- a) By covalently attaching it to a solid support
- a) In a buffer compatible with the immobilized ligand and the biomolecule of interest
- d) All of the above
- d) All of the above
- a) Yes
- a) Neutral or slightly alkaline buffer
- b) Sepharose beads
- a) Protein A or G
- d) All of the above
- d) All of the above
- c) Sample preparation, Binding, Washing, Elution
