#research methods

Animal Tissue Culture 3

Selective lysis of dividing cells can be achieved by:

Selective lysis of dividing cells while sparing quiescent population can be accomplished using bromodeoxyuridine mediated photosensitization strategy. Cells pulsed with BrdU during S phase incorporate halogenated analog into newly synthesized DNA. Subsequent exposure to bright visible light or near-UV in presence of DNA binding dyes such as Hoechst 33258 sensitizes BrdU-substituted DNA to photolysis where carbon-bromine bond breaks generating uracilyl radicals causing strand breaks overwhelming repair and triggering apoptosis. Non-dividing cells lacking BrdU incorporation remain resistant to same light dose. This suicide approach enriches for label-retaining slow-cycling stem cells and enables purification of G0 fraction for functional assays. MTT assay alone measures viability without selective elimination, caspase induction causes widespread death irrespective of proliferative status, and chromium-51 release assay monitors cytotoxic T lymphocyte mediated membrane lysis quantifying target cell killing but not specifically targeting S phase cells. Hence BrdU plus light provides unique tool for functional separation based on DNA replication status in heterogeneous cultures and developmental systems.

Ref: Poot et al J Histochem Cytochem 1991 BrdU photolysis selective lysis dividing cells; Thermo Fisher BrdU labeling followed by light selective killing.

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Protein-Protein Interaction

BiFC (Bimolecular Fluorescence Complementation) detects:

Bimolecular Fluorescence Complementation is a genetic technique to visualize protein-protein association in living cells. Candidate proteins are fused to non-fluorescent N- and C-terminal fragments of a fluorescent protein such as YFP or Venus. Individually fragments are non-fluorescent. When fused proteins interact, fragments are brought into proximity to refold into functional fluorophore, emitting fluorescence detectable by microscopy or flow cytometry. Irreversible complementation reports localization of interaction. It does not measure RNA cleavage, phosphorylation per se, or mismatch repair but specifically protein-protein interaction.

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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Protein-Protein Interaction

Which of the following is not a protein interaction method?

Techniques to map physical protein associations include co-immunoprecipitation that isolates native complexes via specific antibodies, fluorescence resonance energy transfer that detects proximity under 10 nm in live cells, phage display linking phenotype to genotype, yeast two-hybrid, surface plasmon resonance, and affinity pull-downs. SDS-PAGE is a fundamental analytical separation method that denatures proteins with sodium dodecyl sulfate and resolves them by molecular weight under electric field. It provides size information but alone does not report interaction without further methods like far-western or immunoblotting for detection.

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

What type of microarray uses cell lysates?

Protein microarrays are classified by immobilized material and applications. Functional arrays spot purified proteins to test biochemical activities. Analytical arrays immobilize antibodies to measure protein abundance. Reverse-phase arrays invert format by using complex biological samples such as cell lysates, tissue extracts, or fractionated proteomes spotted on chip. These spots are then probed with specific antibodies or ligands. This format enables parallel profiling of post-translational modifications, signaling states, or biomarkers across many samples with limited volume, contrasting drug or antibody-only arrays.

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 a research problem, a student finds proteins A-D with gel filtration, A-C with Co-IP, and B with Y2H. What is the bes

Gel filtration separates native protein complexes by hydrodynamic size; co-elution suggests residence in same large complex but does not distinguish direct versus bridged association. Co-immunoprecipitation from lysates preserves multiprotein assemblies, confirming co-complex membership. Yeast two-hybrid tests binary, direct physical interaction inside nucleus because reporter activation requires intimate contact between bait and prey fusions independent of other yeast proteins. When protein B uniquely appears positive in two-hybrid while others co-purify only, it indicates B is the direct interactor contacting bait, while A, C, D likely associate via B.

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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Hybridization

Reverse-phase microarrays are unique because:

Reverse-phase protein microarrays represent an inverted configuration compared to conventional forward arrays. Instead of printing capturing agents, complex biological samples such as tissue lysates, cell extracts, or body fluids are directly spotted onto slide, with each spot representing entire proteome of one sample. Subsequent detection uses specific antibodies against target protein or modified residue, enabling quantification of protein expression or phosphorylation across many clinical samples simultaneously. This format does not print antibodies as capture, does not rely on whole cells alone, and does not require TEV protease, which belongs to TAP methodology.

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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Electrophysiological methods

Which method allows studying ligand-gated ion channels?

Ligand-gated ion channels such as nicotinic acetylcholine, GABA-A, glycine and ionotropic glutamate receptors open upon neurotransmitter binding, producing rapid postsynaptic currents. Studying them requires ability to apply agonist at defined concentrations while monitoring transmembrane current. Patch-clamp in outside-out or whole-cell mode permits rapid solution exchange via perfusion systems, recording dose-dependent activation, desensitization, single-channel conductance and allosteric modulation with pharmacological precision. EEG records summed population potentials, CT images anatomy using X-rays, MRI uses proton resonance, none provide controlled ligand application and channel current resolution.

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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