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#real-time detection

2 public questions tagged with this topic.

Protein-Protein Interaction

Which interaction is label-free and measured in real-time?

Ideal interaction analysis provides association rate, dissociation rate, and equilibrium dissociation without labeling proteins, avoiding artefacts from fluorophores or radioisotopes and enabling kinetic quantification. Surface Plasmon Resonance achieves this by optical detection of refractive index changes upon analyte-ligand binding on gold sensor chip. It operates continuously, generating sensorgrams in real-time with high sensitivity. EMSA requires labeled nucleic acid, FRET needs fluorescent fusions, BiFC relies on split fluorescent protein complementation. SPR therefore provides unbiased, label-free quantification of protein-protein binding specificity and affinity in real-time.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Protein-Protein Interaction

In SPR, what causes change in resonance units?

Surface Plasmon Resonance monitors binding kinetics in real-time on a gold-coated sensor chip without labels. Ligand is covalently immobilized on dextran matrix, analyte flows over. Incident polarized light excites surface plasmons; resonance angle depends on refractive index near surface. When analyte binds ligand, local mass concentration increases, altering refractive index and shifting resonance angle, recorded as resonance units versus time. Temperature fluctuations or UV are minimized and electric fields are not relevant; angle shift predominantly reflects binding-induced refractive index change.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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