Chapter 8: Analyzing Cells, Molecules, and Systems

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Tissue dissociation

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44 Terms

1

Tissue dissociation

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

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2

Cell separation

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

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3

Primary cell culture

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

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4

Immortalized cell lines

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

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5

Cell culture conditions

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

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Cell adhesion

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

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Cell proliferation

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

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8

Contamination control

Maintaining sterility is crucial to avoid contamination in cell culture.

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9

Cell lysis

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

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14

Fractionation

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

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15

Protein extraction

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

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16

Salting out

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

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17

Solvent extraction

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

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18

Chromatography

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

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19

Affinity chromatography

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

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Ion-exchange chromatography

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

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Size-exclusion chromatography

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

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Hydrophobic interaction chromatography

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

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High-performance liquid chromatography

Utilizes advanced liquid chromatography techniques for higher resolution and efficiency.

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Protein analysis

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

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Protein refolding

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

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Protein quantification

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

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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.

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Western blotting

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

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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.

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Mass Spectrometry

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

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Protein-Protein interactions

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

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Proteomics

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

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DNA extraction

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

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Polymerase chain reaction

PCR amplifies specific DNA sequences in vitro.

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Gel electrophoresis

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

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DNA sequencing

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

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DNA cloning

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

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Restriction enzymes

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

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39

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.

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Transcriptomics

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

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Reporter assays

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

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Functional Assays

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

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statistical analysis

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

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Systems biology

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

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