Q.18 Which type of polyacrylamide gel can be used for analyzing the four different proteins listed below?
Protein P: 60kDa, pI4
Protein Q: 45 kDa , pI 8
Protein R: 60 kDa , pI 6
Protein S: 45kDa, pI7.5
(A) 20% gel, pH4 −7
(B) 20% gel, pH3 −10
(C) 12% gel, pH3 − 10
(D) 12%gel, pH4 −7
The correct answer is (C) 12% gel, pH 3–10. This option provides optimal separation for the four proteins (P: 60 kDa, pI 4; Q: 45 kDa, pI 8; R: 60 kDa, pI 6; S: 45 kDa, pI 7.5) in native polyacrylamide gel electrophoresis (native PAGE), where separation depends on both molecular weight (MW) and isoelectric point (pI) based on net charge at the buffer pH.
Protein Properties
Proteins P and R share 60 kDa MW but differ in pI (4 vs. 6), while Q and S share 45 kDa but differ in pI (8 vs. 7.5). In native PAGE, migration depends on size (smaller proteins move faster) and charge (more negative net charge at gel pH speeds migration toward the anode).
Gel Percentage Analysis
-
12% gels suit 20-100 kDa proteins like these (45-60 kDa), offering good pore size for resolution without restricting larger ones.
-
20% gels target <30-45 kDa; they compress 45-60 kDa bands poorly, hindering separation.
pH Range Evaluation
pH must ensure all proteins have net negative charge (pH > pI) for anodic migration and distinct mobilities.
-
pH 3-10 covers all pI values (4-8), allowing charge differences (e.g., P most negative at low pH, Q least).
-
pH 4-7 fails for Q (pI 8 >7, net positive, no migration).
Option Breakdown
| Option | Gel % Suitability | pH Coverage | Why Incorrect/Correct |
|---|---|---|---|
| (A) 20% gel, pH 4-7 | Poor (too dense for 45-60 kDa) | Fails Q (positive charge) | Cannot resolve sizes or migrate all proteins |
| (B) 20% gel, pH 3-10 | Poor (compresses bands) | Good (all negative) | Size resolution inadequate |
| (C) 12% gel, pH 3-10 | Good (optimal pores) | Good (differential charges) | Resolves both size (45 vs 60 kDa) and charge differences |
| (D) 12% gel, pH 4-7 | Good | Fails Q | Q does not migrate |
Polyacrylamide gel electrophoresis (PAGE) is essential for separating proteins by size and charge in molecular biology, especially for CSIR NET aspirants tackling native PAGE questions on 45-60 kDa proteins with pI values from 4 to 8. Selecting the right gel percentage and pH range ensures clear resolution of bands like Protein P (60 kDa, pI 4), Q (45 kDa, pI 8), R (60 kDa, pI 6), and S (45 kDa, pI 7.5).
Key Principles of Native PAGE
Native PAGE preserves protein structure, separating based on MW and net charge at gel pH (negative charge drives anodic migration when pH > pI). For mixed pI (acidic to basic), broad pH 3-10 buffers provide differential migration without immobilizing any protein.
Ideal Gel Percentage for 45-60 kDa
-
12% polyacrylamide offers pore sizes perfect for 20-100 kDa, resolving close MW like 45 vs 60 kDa.
-
Avoid 20%: Suits <45 kDa; larger proteins barely enter, causing poor separation.
pH Range: Why 3-10 Wins
pH 3-10 spans all pI 4-8, ensuring net negative charges with varying strengths (P fastest, Q slowest). Narrow pH 4-7 leaves basic pI 8 positive, preventing migration.
