Chapter 8: Analyzing Cells, Molecules, and Systems

Tissue dissociation

Tissue dissociation

Tissues can be dissociated to obtain a single-cell suspension for cell culture.

Cell separation

If a specific cell population is desired, cells can be separated based on their physical or molecular properties.

Primary cell culture

Primary cells are isolated directly from tissues and have a limited lifespan in culture.

Immortalized cell lines

Immortalized cell lines are derived from primary cells but have undergone genetic modifications to overcome replicative senescence.

Cell culture conditions

Culture conditions include temperature, humidity, and a specific gas composition (typically 5% CO2 and 95% air).

Cell adhesion

Culture vessels can be coated with extracellular matrix proteins such as collagen, fibronectin, or gelatin to enhance cell adhesion.

Cell proliferation

Cells in culture proliferate and expand over time, forming a monolayer or three-dimensional structures.

Contamination control

Maintaining sterility is crucial to avoid contamination in cell culture.

Cell lysis

Cells are lysed to release their contents, including the target protein of interest.

Fractionation

Cell lysate is subjected to fractionation techniques to separate cellular components based on their size, charge, or solubility.

Protein extraction

Proteins can be extracted from cellular fractions using various techniques, depending on their physicochemical properties.

Salting out

Precipitation of proteins by adding high concentrations of salts (e.g., ammonium sulfate).

Solvent extraction

Partitioning of proteins based on their solubility in organic solvents or detergents.

Chromatography

Separation of proteins based on their affinity for specific ligands or physical properties.

Affinity chromatography

Exploits specific interactions between the target protein and an immobilized ligand (e.g., antibody, metal ions, or receptor).

Ion-exchange chromatography

Separates proteins based on their net charge and affinity for charged resin.

Size-exclusion chromatography

Separates proteins based on their size and shape, allowing for the removal of contaminants.

Hydrophobic interaction chromatography

Separates proteins based on their hydrophobicity, utilizing the interaction between the protein and a hydrophobic resin.

High-performance liquid chromatography

Utilizes advanced liquid chromatography techniques for higher resolution and efficiency.

Protein analysis

Throughout the purification process, protein fractions are analyzed to assess purity and quantity.

Protein refolding

If a purified protein has lost its native conformation and activity during purification, refolding may be required.

Protein quantification

Protein quantification methods determine the concentration of proteins in a sample.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE separates proteins based on their size. Proteins are denatured and coated with SDS, a detergent that imparts a negative charge to the proteins.

Western blotting

Western blotting detects and characterizes specific proteins within a complex mixture.

Enzyme-Linked Immunosorbent Assay

ELISA detects and quantifies specific proteins using antibodies. The target protein is immobilized on a solid surface, such as a microplate.

Mass Spectrometry

Mass spectrometry analyzes proteins by measuring their mass-to-charge ratio.

Protein-Protein interactions

Studying protein-protein interactions helps understand protein function and cellular processes.

Proteomics

Proteomics involves the large-scale analysis of proteins and their modifications.

DNA extraction

DNA extraction is the first step in analyzing and manipulating DNA.

Polymerase chain reaction

PCR amplifies specific DNA sequences in vitro.

Gel electrophoresis

Gel electrophoresis separates DNA fragments based on their size and charge.

DNA sequencing

DNA sequencing determines the order of nucleotide bases in a DNA molecule.

DNA cloning

DNA cloning involves the replication of a specific DNA fragment and its insertion into a vector for replication in a host organism.

Restriction enzymes

Restriction enzymes, also known as restriction endonucleases, cleave DNA at specific recognition sites.

CRISPR-Cas 9 genome editing

CRISPR-Cas9 is a revolutionary genome editing tool. It utilizes a guide RNA (gRNA) to target specific DNA sequences, and the Cas9 nuclease introduces precise cuts at the target site.

Transcriptomics

Transcriptomics investigates gene expression patterns by analyzing the complete set of RNA molecules (transcriptome) in a cell or tissue.

Reporter assays

Reporter assays allow the measurement of gene expression levels and promoter activity.

Functional Assays

Functional assays assess the biological activity of genes or gene products.

statistical analysis

Statistical analysis is crucial for evaluating the significance of experimental results and validating mathematical models.

Systems biology

Systems biology aims to understand biological systems as a whole by integrating experimental data with mathematical modeling.