Q.67 Two proteins X (Mol. wt. 20 kD, pI 6.3) and Y (Mol. wt. 90 kD, pI 6.4) can be separated by which of the following techniques?
A. Gel filtration
B. Ion-exchange chromatography
C. Isoelectric focussing
D. SDS-PAGE
Choose the correct answer from the options given below.
- A and B only
- A and D only
- B and C only
- B and D only
Answer: A and D only
Proteins X (20 kDa, pI 6.3) and Y (90 kDa, pI 6.4) differ significantly in molecular weight but have nearly identical pI values, allowing separation by size-based techniques.
Option Analysis
Gel Filtration (A)
Gel filtration (size-exclusion chromatography) separates proteins by hydrodynamic volume/molecular weight; smaller X (20 kDa) elutes later than larger Y (90 kDa) as it penetrates more gel pores. Effective regardless of similar pI values.Ion-Exchange Chromatography (B)
Relies on charge differences at a given pH; pI 6.3 vs. 6.4 is too close (~0.1 unit) for reliable separation, as both proteins have similar net charges near their pI.Isoelectric Focusing (C)
Separates by exact pI in a pH gradient; ΔpI of 0.1 is typically insufficient for baseline resolution without ultra-high-resolution setups.SDS-PAGE (D)
Denatures proteins with SDS (proportional mass/charge) and separates by size in an electric field; 20 kDa vs. 90 kDa yields clear band separation on the gel.Protein separation by molecular weight and pI is essential in biochemistry for purifying proteins like X (20 kDa, pI 6.3) and Y (90 kDa, pI 6.4). With similar pI values but stark size differences, gel filtration and SDS-PAGE excel, while ion-exchange and isoelectric focusing fail due to minimal charge variance. This technique selection is key for competitive exams testing purification strategies.
Technique Breakdown
Size-based methods dominate when pI values are close:
Technique Basis Can Separate X & Y? Reason Gel Filtration Size (20 vs 90 kDa) Yes Smaller protein elutes later Ion-Exchange Charge (pI 6.3 vs 6.4) No ΔpI too small Isoelectric Focusing pI exactly No Resolution insufficient SDS-PAGE SDS-denatured size Yes Clear band separation Practical Applications
In proteomics workflows, combine gel filtration for native purification with SDS-PAGE for analysis. For protein separation techniques molecular weight pI, always match method to property differences—size here trumps subtle pI shifts.
