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8. Analyzing Cells, Molecules and Systems
ISOLATING CELLS AND GROWING THEM IN CULTURE
- The first step in isolating individual cells is to disrupt the extracellular matrix and cell–
cell junctions. A tissue sample can be treated with proteolytic enzymes to digest
proteins in the extracellular matrix and with agents that chelate Ca2+ on which cell–cell
- Fluorescence-activated cell sorter is one of the most sophisticated cell-separation
techniques that use an antibody coupled to a fluorescent dye to label specific cells and
separate them from the others. The antibody chosen recognizes an epitope present
on the surface of a specific type of cell.
- Primary cultures are prepared directly from the tissues of an organism – these cells
eventually die due to a process called replicative cell senescence.
- Immortalized cell lines can be cultures indefinitely. These are transformed cells that
were modified to induce activity of the telomerase and/or to express certain cancer-
- Monoclonal antibodies are produced by a certain type of transformed cancer cell called
hybridoma. These antibodies are used to detect and purify cellular proteins, as well as
to diagnose and treat diseases.
- Highly purified cell-free systems are needed to determine the molecular details of
complex cellular processes.
- Ultracentrifuges rotate extracts of broken cells (homogenates of whole cells) at high
speed to separate them according to their size and density.
- Velocity sedimentation separates subcellular components at different speeds
according to their size and shape when layered over a solution containing sucrose.
Equilibrium sedimentation separates subcellular components in a gradient solution
until they reach a position where their density matches the gradient.
- Column chromatography is a method of separation in which a mixture of proteins in
solution is passed through a column containing a porous solid matrix. Different proteins
are retarded to different extents by their interaction with the matrix and collected
separately. Depending on the choice of matrix, proteins can be separated according
to their charge (ion-exchange chromatography), hydrophobicity (hydrophobic
chromatography), size (gel-filtration chromatography) and ability to bind other
molecules (affinity chromatography).
- Immunoprecipitation is another method to
purify proteins that is related to affinity
chromatography. In this method, specific
antibodies that recognize the protein of
interest are attached to small beads and a
small quantity of the antibody-coated beads
is added to a protein extract in a test tube
- SDS polyacrylamide-gel electrophoresis (SDS-PAGE) is a method used to separate
protein in a matrix according to their size (separation in one dimension). In this method,
all proteins will have a negative charge due to binding of SDS that is negatively
- Two-dimensional gel electrophoresis provides two different separations: by net charge
and by size. Protein net charge is separated in a pH gradient by a procedure called
isoelectric focusing; later SDS is mixed with the sample – to provide negative charge
to all proteins - and current is applied to separate proteins by size.