56. Affinity chromatography is a method that can be used to purify cell surface receptors, while they retain their hormone binding ability.
A ligand (hormone) for a receptor of interest is chemically linked to polystyrene beads.
A solubilized preparation of membrane proteins is passed over a column containing these beads.
Only the receptor binds to the beads.
When an excess of the ligand (hormone) is poured through the column after the receptor binding step, what do you expect will occur?
A. The ligand will attach to those beads that have the receptor and remain on the column
B. The ligand will cause the receptor to be displaced from the beads and eluted out
C. The ligand will attach to the bead instead of the receptor
D. The ligand will cause the bead to lose its affinity by changing shape
Answer: (B) The ligand will cause the receptor to be displaced from the beads and eluted out
In affinity chromatography, adding excess soluble ligand (hormone) after receptor binding creates competition with the immobilized ligand on beads, displacing the receptor for purification.
Option Analysis
(A) Ligand Attaches to Beads with Receptor
Incorrect. The beads already have covalently linked ligand; excess soluble ligand cannot bind to beads and instead competes for receptor binding sites.
(B) Ligand Displaces Receptor from Beads
Correct. Competitive elution uses high concentrations of free ligand to outcompete immobilized ligand, breaking receptor-bead interactions so receptor elutes in solution while retaining activity.
(C) Ligand Attaches to Bead Instead of Receptor
Wrong. Bead ligands are chemically fixed (covalent bonds to polystyrene/agarose); soluble ligand cannot displace or attach to beads.
(D) Ligand Changes Bead Shape/Affinity
Incorrect. Beads maintain structural integrity; elution relies on equilibrium shift, not physical bead alteration.
Affinity chromatography ligand displaces receptor through competitive elution when excess hormone is added after binding, a key purification step for cell surface receptors in biochemistry.
Competitive Elution Mechanism
Solubilized membrane proteins pass over hormone-linked polystyrene beads; only target receptor binds specifically.
Excess free ligand (hormone) is then applied, competing with immobilized ligand (higher effective concentration in solution) to shift equilibrium: Receptor + free ligand ⇌ Receptor-ligand (soluble), eluting pure receptor.
This biospecific method preserves hormone-binding activity unlike harsh pH/denaturant elution.
Affinity Chromatography Steps Comparison
| Step | Action | Outcome |
| Loading | Membrane proteins over ligand-beads | Receptor binds; others wash through |
| Washing | Buffer removes weak binders | High specificity achieved |
| Elution | Excess ligand added | Receptor displaced & eluted |
| Regeneration | Buffer re-equilibrates beads | Column reusable |
This table shows why competitive elution with excess ligand is standard for receptor purification in GATE questions.
GATE Exam Applications
Tests understanding of reversible biospecific interactions; pairs with prior insulin receptor questions for technique mastery.
