18. The movement of proteins within the nucleus can be determined by:
A. Electron microscopy
B. Fluorescence recovery after photobleaching
C. Fluorescent in situ hybridization
D. Confocal light microscopy
Fluorescence Recovery After Photobleaching (FRAP) directly measures protein mobility by tracking fluorescent recovery in photobleached nuclear regions.
Correct Answer
B. Fluorescence recovery after photobleaching. FRAP quantifies diffusion rates and binding kinetics of nuclear proteins like transcription factors.
Option Breakdown
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A. Electron microscopy: Incorrect. Fixed/static imaging; cannot monitor dynamic protein movement in live nuclei.
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B. Fluorescence recovery after photobleaching: Correct. Bleaches GFP-fusion proteins in nuclear ROI, measures recovery rate via diffusion from unbleached areas.
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C. Fluorescent in situ hybridization: Incorrect. Fixed cell technique for DNA/RNA localization; no dynamic movement data.
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D. Confocal light microscopy: Incorrect. Provides optical sectioning but doesn’t measure mobility; requires FRAP protocol for movement quantification.
Fluorescence recovery after photobleaching nuclear proteins reveals diffusion coefficients (D), binding residence times, and mobile/immobile fractions within live cell nuclei.
FRAP Protocol: Express GFP-histone/transcription factor → 488 nm bleach → time-lapse imaging → fit recovery curve → extract D = r<sup>2</sup>/4t<sub>1/2</sup>.
Technique Comparison
| Technique | Live Cell | Measures Movement | Nuclear Proteins | GATE Answer |
|---|---|---|---|---|
| FRAP | Yes | Diffusion + binding | Perfect | Correct |
| Electron Microscopy | No | Static | Fixed only | No dynamics |
| FISH | No | Localization | DNA/RNA | Static |
| Confocal | Yes | Imaging only | No kinetics | Insufficient |
Mobile fraction = recovered fluorescence; immobile = chromatin-bound proteins.
Nuclear Applications
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Transcription factors: Residence time ~seconds (diffusion-dominated)
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Histones: >90% immobile fraction
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RNA Pol II: Scan rapidly, pause at promoters
Exam Key: FRAP = movement quantification. Confocal = static imaging. FISH/EM = fixed specimens.
