Advanced Biochemical and Molecular Biological Methods

A collection of vocabulary flashcards covering cell culture models, viability assays, nucleic acid sequencing technologies, and molecular interaction analysis methods derived from lecture notes.

Cell culture

The cultivation of cells outside a living organism within controlled laboratory environments to study physiological and pathological mechanisms.

2D Cell Culture

A model where cells grow as a monolayer on a flat surface, utilized for its technical simplicity, reproducibility, and high visibility under microscopy.

3D Cell Culture

A cultivation system designed to better represent in vivo microarchitecture, cell morphology, and communicative metabolic activity.

Sterility

The fundamental principle of cell cultivation requiring aseptic techniques and sterile consumables to prevent microbiological or chemical contamination.

Incubator Environment

The physical setting providing controlled temperature (such as $37\,^∘\text{C}$ for mammals) and $\text{pH}$ stabilization, frequently through a $5\%$ $\text{CO}_2$ buffer system.

Trypsin

An enzyme often used during the passaging of adherent cultures to facilitate the release of cells from the growth surface.

Confluence

A practical criterion expressing the percentage of the growth surface covered by cells, used to determine timing for subculturing or experimentation.

Trypan Blue Exclusion Test

A membrane-integrity method where viable cells exclude the dye while cells with damaged membranes stain blue.

Propidium Iodide

A fluorescent dye that penetrates only damaged plasma membranes to intercalate into DNA, producing a red signal often used in flow cytometry.

MTT Assay

A colorimetric metabolic assay based on the reduction of tetrazolium salt to insoluble formazan crystals by mitochondrial dehydrogenases.

Resazurin Reduction Assay

A sensitive, non-toxic viability test where a blue dye is converted to pink fluorescent resorufin by metabolically active cells.

Maxam-Gilbert Sequencing

A first-generation chemical sequencing approach that uses specific chemical treatments to create breaks at purine or pyrimidine bases.

Sanger Sequencing

A chain-termination method requiring $\text{DNA}$ polymerase and modified dideoxynucleotides ($\text{ddNTPs}$) to stop $\text{DNA}$ synthesis at specific bases.

Illumina Sequencing

An industry-standard second-generation platform utilizing sequencing by synthesis with reversible terminators and bridge amplification.

Ion Torrent

A sequencing technology that records signals based on the release of hydrogen ions ($H^+$) during nucleotide incorporation.

SMRT Technology

Single-Molecule Real-Time sequencing where $\text{DNA}$ polymerase is fixed within zero-mode waveguides to detect fluorescently labeled nucleotides.

Oxford Nanopore Sequencing

A third-generation method that measures disruptions in ionic current as nucleic acids pass through a protein pore to determine sequential motifs.

CRISPR-Cas

A programmable genome-editing tool where a guide $\text{RNA}$ directs a nuclease to create targeted $\text{DNA}$ breaks for repair-mediated modification.

$$K_d$$

The dissociation constant, where a lower value signifies a higher binding affinity between a protein and its ligand.

Chromatin Immunoprecipitation (ChIP)

A method used to identify $\text{DNA}$-protein interactions by stabilizing complexes through cross-linking and isolating them with specific antibodies.

EMSA

The Electrophoretic Mobility Shift Assay, which identifies protein-nucleic acid complexes based on their reduced migration speed through a gel compared to free probes.

Yeast Two-Hybrid System

An in vivo screening method based on the reconstitution of the $\text{GAL4}$ transcription factor to detect protein-protein interactions via reporter gene expression.

Differential Scanning Fluorimetry (DSF)

A thermal shift assay that detects protein-ligand interactions by monitoring changes in the melting temperature ($T_m$) using a hydrophobic fluorescent dye.

Isothermal Titration Calorimetry (ITC)

A gold-standard technique that provides a complete thermodynamic description of an interaction by measuring heat changes during stepwise ligand injections.

Surface Plasmon Resonance (SPR)

A label-free optical biosensor method that monitors the kinetics of association and dissociation by detecting refractive index changes near a metal surface.

Biolayer Interferometry (BLI)

A real-time interaction analysis method that measures shifts in the optical interference pattern of light reflected from a biosensor tip.

Proteoliposome

An artificial system where a purified transporter protein is integrated into a lipid bilayer to study its function in a defined environment.

High-Resolution Respirometry

A technique for analyzing mitochondrial bioenergetics by measuring oxygen consumption, often following a specific substrate-uncoupler-inhibitor titration (SUIT) protocol.