Q.36 A mixture of the following purified proteins, IgG, IgM and Fab fragment of
immunoglobulins, is separated using gel filtration chromatography. The order of elution of
these proteins (first to last) is
(A)Fab fragment, IgM and IgG
(B) IgM, IgG and Fab fragment
(C) Fab fragment, IgG and IgM
(D) IgG, IgM and Fab fragment
Gel filtration chromatography separates proteins by size, with larger molecules eluting first. In a mixture of IgG, IgM, and Fab fragment, the correct elution order is IgM, followed by IgG, then Fab fragment.
Principle of Separation
Gel filtration, or size-exclusion chromatography, uses porous beads where larger proteins cannot enter pores and elute faster in the void volume. Smaller proteins diffuse into pores, increasing their path length and delaying elution. Molecular weights dictate order: IgM (~900 kDa), IgG (~150 kDa), Fab (~50 kDa).
Protein Sizes
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IgM: Pentameric antibody, largest at 900 kDa, elutes first as it excludes from pores.
-
IgG: Monomeric, 150 kDa, elutes after IgM but before smaller fragments.
-
Fab Fragment: Antigen-binding part of IgG, ~50 kDa, smallest and elutes last.
Option Analysis
| Option | Elution Order | Correct? | Reason |
|---|---|---|---|
| (A) Fab, IgM, IgG | Smallest first | No | Violates size-exclusion; largest elutes first . |
| (B) IgM, IgG, Fab | Largest to smallest | Yes | Matches decreasing size: 900 > 150 > 50 kDa . |
| (C) Fab, IgG, IgM | Small, medium, large | No | Reverse of actual principle . |
| (D) IgG, IgM, Fab | Medium, large, small | No | IgM larger than IgG, so elutes before . |
