Isoelectric focusing principle — Proteins are separated primarily based on what compositional/physicochemical property?
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ARelative content of positively charged residues only
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BRelative content of negatively charged residues only
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CSize (molecular weight)
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DOverall balance of positive and negative residues reflected in isoelectric point (pI)
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EHydrophobicity alone in detergent micelles
Answer
Correct Answer: Overall balance of positive and negative residues reflected in isoelectric point (pI)
Explanation
Introduction:Isoelectric focusing (IEF) separates proteins along a fixed pH gradient until each focuses at its isoelectric point, the pH where net charge equals zero. Understanding pI connects sequence composition to separations in proteomics workflows.
Given Data / Assumptions:
- A stable pH gradient exists across the gel or capillary.
- Proteins migrate under an electric field until net charge is zero.
- pI depends on the balance of acidic and basic residues and their pKa values.
Concept / Approach:Because migration stops when net charge is zero, proteins resolve sharply at pI values. This is complementary to SDS–PAGE (size-based) and can be combined in 2D electrophoresis (IEF in the first dimension, SDS–PAGE in the second).
Step-by-Step Solution:
1) Apply electric field → proteins move according to instantaneous charge at local pH.2) Each protein reaches the pH where positive and negative charges balance (pI) → net charge 0.3) Focusing occurs because any displacement from pI restores charge and drives the protein back.Verification / Alternative check:Mixing pI markers with samples confirms gradient integrity and focusing performance; spots align with expected pI values in 2D maps.
Why Other Options Are Wrong:
a,b) pI reflects both positive and negative residues, not one class alone.c) Size is not the IEF basis; that is SDS–PAGE.e) Hydrophobicity affects chromatography with detergents, not IEF.Common Pitfalls:Assuming mobility ceases for all proteins at the same pH; overlooking ampholyte gradient stability; confusing IEF with charge-based native PAGE lacking a fixed gradient.
Final Answer:Separation is by isoelectric point (pI), reflecting the overall balance of positive and negative residues.