Cell Biologist Tools Guide

© 2020 Elsevier Inc. All rights reserved.
Chapter 1
Tools of the Cell Biologist

© 2020 Elsevier Inc. All rights reserved. 2
Figure 1–1 Structural features of animal cells. Summary of the functions of cellular organelles. Mitochondria: (1) site of the Krebs (citric
acid) cycle, produce ATP by oxidative phosphorylation; (2) can release apoptosis-initiating proteins, such as cytochrome c. Cytoskeleton:
made up of microfilaments, intermediate filaments, and microtubules; governs cell movement and shape. Centrioles: components of the
microtubule-organizing center. Plasma membrane: consists of a lipid bilayer and associated proteins. Nucleus: contains chromatin (DNA
and associated proteins), gene regulatory proteins, and enzymes for RNA synthesis and processing. Nucleolus: the site of ribosome RNA
synthesis and ribosome assembly. Ribosomes: sites of protein synthesis. Rough ER, Golgi apparatus, and transport vesicles: synthesize
and process membrane proteins and export proteins. Smooth ER: synthesizes lipids and, in liver cells, detoxifies cells. Lumen: Ca2 +
reservoir. Clathrin-coated pits, clathrin-coated vesicles, and early and late endosomes: sites for uptake of extracellular proteins and
associated cargo for delivery to lysosomes. Lysosomes: contain digestive enzymes. Peroxisomes: cause β-oxidation of certain lipids
(e.g., very long chains of fatty acids). (Modified from Freeman S. Biological Science, 1st ed. Upper Saddle River, NJ: Prentice Hall, 2002.)

Figure 1–2 Comparison of the lens systems in a light microscope and a transmission electron microscope. In a light microscope (left),
light is focused on the sample by the condenser lens. The sample image is then magnified up to 1000 times by the objective and ocular
lenses. In a transmission electron microscope (right), magnets serve the functions of the condenser, objective, and ocular (projection)
lenses, focusing the electrons and magnifying the sample image up to 250,000 times. (Modified from Alberts B, et al. Molecular Biology of
the Cell, 4th ed. New York, NY: Garland Science, 2002.)

Figure 1–3 Light passing through a sample is diffracted, producing edge effects. When light waves pass near the edge of a barrier, they
bend and spread at oblique angles. This phenomenon is known as diffraction. Diffraction produces edge effects because of constructive
and destructive interference of the diffracted light waves. These edge effects limit the resolution of the image produced by microscopic
magnification. (Modified from Alberts B, et al. Molecular Biology of the Cell, 4th ed. New York, NY: Garland Science, 2002.)

Figure 1–4 Numerical aperture (NA). The NA of a microscopic objective is defined as n sin α, where n is the refractive index of the
medium between the sample and the objective lens (air), and α, the angular aperture, is the half angle of the cone of light entering the
objective lens from a focal point in the sample. Objective lenses with increasingly high NA values (A, B, and C) collect increasingly more
light from the sample. (Modified from http://www.microscopyu.com/articles/formulas/formulasna.html.)

Figure 1–5 Fluorescence microscopy. (A) Optical layout of a fluorescence microscope. Incident light tuned to excite the fluorescent
molecule is reflected by a dichroic mirror, and then focused on the sample; fluorescent light (longer wavelength than excitation light)
emitted by the sample passes through the dichroic mirror for viewing. (B) Immunofluorescent micrograph of a human skin fibroblast,
stained with fluorescent antiactin antibody. Cells were fixed, permeabilized, and then incubated with fluorescein-coupled antibody.
Unbound antibody was washed away before viewing. ([A] Modified from Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D,
Darnell J. Molecular Cell Biology, 4th ed. New York, NY: W.H. Freeman, 2000; [B] courtesy E. Lazerides.)

Figure 1–6 Structure of an antibody molecule. An antibody molecule consists of two identical heavy chains, plus two identical light
chains. The disulfide-bonded, carboxyl-terminal halves of the heavy chains (the “tail” of the antibody) are jointly called the Fc domain; the
two arms, which bind antigens at their tips, are called the Fab domains. Because all immunoglobulins are modified by the attachment of
carbohydrate, they are examples of a type of protein termed a glycoprotein. The immunoglobulin shown here is an IgG molecule; class M,
A, and E immunoglobulins are roughly similar, except IgM and IgE have larger Fc domains. The different immunoglobulin classes are also
glycosylated at different sites. (Modified from Parham P. The Immune System, 2nd ed. New York, NY: Garland Publishing, 2005.)

Figure 1–7 Monoclonal antibodies. (1) Myeloma cells are fused with antibody-producing cells from the spleen of an immunized mouse.
(2) The mixture of fused hybridoma cells together with unfused parent cells is transferred to a special growth medium (HAT medium) that
selectively kills the myeloma parent cells; unfused mouse spleen cells eventually die spontaneously because of their natural limited
proliferation potential. Hybridoma cells are able to grow in HAT medium and have the unlimited proliferation potential of their myeloma
parent. (3) After selection in HAT medium, cells are diluted and individual clones growing in particular wells are tested for production of
the desired antibody. (Modified from Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL, Darnell J. Molecular
Cell Biology, 5th ed. New York, NY: W.H. Freeman, 2004.)

Figure 1–8 Column chromatography. A porous column of beads equilibrated in a particular solvent is prepared, and a sample containing
a mixture of proteins is applied to the top of the column. The sample is then washed through the column, and the column eluate is
collected in a succession of test tubes. Because of the properties of the beads in the column, proteins with different properties elute at
varying rates off the column. (Modified from Alberts B, et al. Molecular Biology of the Cell. New York, NY: Garland Science, 2002.)

Figure 1–9 Three types of beads used for column chromatography. (A) The beads may have a positive or a negative charge. (The
positively charged beads shown in the figure might, for example, be derivatized with diethylaminoethyl groups, which are positively
charged at pH 7.) Proteins that are positively charged in a pH 7 buffer will flow through the column; negatively charged proteins will be
bound to the beads and can be subsequently eluted with a gradient of salt. (B) The beads can have cavities or channels of a defined size;
proteins larger than these channels will be excluded from the beads and elute in the “void volume” of the column; smaller proteins of
various sizes will, to varying degrees, enter the beads and pass through them, thereby becoming delayed in their elution from the column.
Such columns, therefore, resolve proteins by size. (C) The beads can be derivatized with a molecule that specifically binds the protein of
interest. In the example shown, it is a substrate (or substrate analog) for a particular enzyme; the beads could also be derivatized with an
antibody to the protein of interest, in which case this would be called immunoaffinity chromatography. (Modified from Alberts B, et al.
Molecular Biology of the Cell. New York, NY: Garland Science, 2002.)

Figure 1–10 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (A) Proteins in the sample are heated with the
negatively charged detergent SDS, which unfolds them and coats them with a uniform negative charge density; disulfide bonds (S―S)
are reduced with mercaptoethanol. (B) The sample is applied to the well of polyacrylamide gel slab, and a voltage is applied to the gel.
The negatively charged detergent-protein complexes migrate to the bottom of the gel, toward the positively charged anode. Small proteins
can move more readily through the pores of the gel, but larger proteins move less readily, so individual proteins are separated by size,
smaller toward the bottom and larger toward the top. (Modified from Alberts B, et al. Molecular Biology of the Cell, 4th ed. New York, NY:
Garland Science, 2002.)

Figure 1–11 Western blotting. (1) Proteins are resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The
gel with the resolved set of proteins is then placed in an apparatus that permits electrophoretic transfer of the proteins from the gel to the
surface of a special paper (e.g., nitrocellulose paper) to which proteins strongly adsorb. (2) After transfer the nitrocellulose sheet is
incubated with an antibody (the “primary” antibody) directed against the protein of interest. (Before this incubation [not shown], the surface
of nitrocellulose paper is “blocked” by incubating it with a nonreactive protein such as casein, to prevent nonspecific binding of the 1°
antibody to the nitrocellulose; this casein block leaves the sample proteins still available for antibody binding.) (3) After washing away
unbound 1° antibody, an enzyme-linked 2° antibody is added, which binds the 1° antibody and (4) can generate a colored product for
detection. (Modified from Lodish H, et al. Molecular Cell Biology, 5th ed. New York, NY: W.H. Freeman, 2004.)

Figure 1–12 Two-dimensional gel electrophoresis. (1) Proteins in the sample are first separated by their isoelectric points in a narrow
diameter tube gel with a fixed pH gradient, by a technique called isoelectric focusing (IEF). This is the “first-dimensional” separation. (2)
The IEF gel is then soaked in SDS and laid on top of a slab SDS polyacrylamide gel for the “second-dimensional” separation of SDS-
PAGE (3), which resolves proteins based on their size. (Modified from Lodish H, et al., Molecular Cell Biology, 5th ed. New York, NY:
W.H. Freeman, 2004.)

Figure 1–13 The structure of green fluorescent protein (GFP). GFP is an 11-strand β-barrel, with an α-helical segment threaded up
through the interior of the barrel. The amino- and carboxyl-terminal ends of the protein are free and do not participate in forming the stable
β-barrel structure. Within an hour or so after synthesis and folding, a self-catalyzed maturation process occurs in the protein, whereby
side chains in the interior of the barrel react with each other and with oxygen to form a fluorophore covalently attached to the through-
barrel α-helical segment, near the center of the β-barrel cavity. (Modified from Ormö M, et al. Science 1995;273:1392–1395.)

Figure 1–14 Fluorescence resonance energy transfer (FRET). (A) The two proteins of interest are expressed in cells as fusion proteins
with either blue fluorescent protein (BFP) (protein X) or GFP (protein Y). Excitation of BFP with violet light results in the emission of blue
fluorescent light by BFP; excitation of GFP with blue light yields green fluorescence. (B) If the two proteins do not bind each other inside
the cell, excitation of the BFP molecule with violet light results simply in blue fluorescence. If, however, (C) the two proteins do bind each
other, they will be close enough to permit resonant energy transfer between the excited BFP molecule and the GFP protein, resulting in
green fluorescence after violet excitation. (Modified from Alberts B, et al. Molecular Biology of the Cell, 4th ed. New York, NY: Garland
Science, 2002.)

Figure 1–15 Laser scanning confocal fluorescence microscopy. (A) Optical layout of a laser scanning confocal microscope. Incident
laser light tuned to excite the fluorescent molecule (green) is reflected off a dichroic mirror and is then guided by two scanning mirrors and
the objective lens to the focal plane to illuminate a spot in a focal plane in the specimen. The scanning mirrors rock back and forth so that
the light sweeps across the specimen in raster fashion. The fluorescence (green) emitted by the fluorescently tagged molecules in the
specimen is then sent back to be captured by a photomultiplier tube; on the way back, it must pass through a pinhole that is confocal with
the specimen focal plane. The confocal pinhole excludes light from out-of-focus focal planes in the specimen. The fluorescence signals
from photomultiplier tube are then sent to a computer that reconstructs the confocal image. (B) An image of fluorescent tubulin in a
fertilized sea urchin egg as viewed with conventional fluorescence microscopy. (C) The same fertilized egg viewed by confocal
microscopy in a focal plane that passes through the equator of the egg. One sees clearly the mitotic spindle apparatus as the egg is
dividing into two daughter cells. ([A] Modified from Lodish H, Berk A, Kaiser C, Krieger M, Bretscher A, Ploegh H, Amon A, Martin C.
Molecular Cell Biology, 8th ed. New York: W.H. Freeman, 2016; [B] and [C] White J, et al. J Cell Biology 1987;105:41–48, by permission
of the Rockefeller University Press.)

Figure 1–16 Superresolution fluorescence microscopy. (A) A confocal image of fluorescently labeled microtubules located near the
periphery of a fibroblast (BSC-1 cells). (B) A corresponding superresolution image of the same microtubules. (From Xiaowei Zhuang:
Huang et al. Science 2008;319:810–813, with permission from AAAS).

Figure 1–17 Protein A–coated gold particles can be used to localize antigen-antibody complexes by transmission electron microscopy
(EM). (A) Protein A is a bacterial protein that specifically binds the Fc domain of antibody molecules, without affecting the ability of the
antibody to bind antigen (the enzyme catalase, in the example shown here); it also strongly adsorbs to the surface of colloidal gold
particles. (B) Anticatalase antibodies have been incubated with a slice of fixed liver tissue, where they bind catalase molecules. After
washing away unbound antibodies, the sample was incubated with colloidal gold complexed with protein A. The electron-dense gold
particles are thereby positioned wherever the antibody has bound catalase, and they are visible as black dots in the electron micrograph.
It is apparent that catalase is located exclusively in peroxisomes. ([A] Modified from Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger
M, Scott MP, Zipursky SL, Darnell J. Molecular Cell Biology, 5th ed. New York, NY: W.H. Freeman, 2004; [B] from Geuze HF, et al. J Cell
Biol 1981;89:653, by permission of the Rockefeller University Press.)

Figure 1–18 Electron microscopic images of negatively stained versus metal-shadowed specimens. A preparation of tobacco rattle virus
was either (A) negatively stained with potassium phosphotungstate or (B) shadowed with chromium. (Courtesy M. K. Corbett.)

Figure 1–19 Procedure for metal shadowing. The specimen is placed in a special bell jar, which is evacuated. A metal electrode is
heated, causing evaporation of metal atoms from the surface of the electrode. The evaporated metal atoms spray over the surface of the
sample, thereby “shadowing” it. (Modified from Karp G. Cell and Molecular Biology, 3rd ed. New York: John Wiley & Sons, 2002.)

Figure 1–20 Cryoelectron microscopy of cytoskeletal filaments, obtained by deep etching. A fibroblast was gently extracted using the
nonionic detergent Triton X-100 (Sigma, St. Louis), which dissolves the surface membrane and releases soluble cytoplasmic proteins, but
has no effect on the structure of cytoskeletal filaments. The extracted cell was then rapidly frozen, deep etched, and shadowed with
platinum, then viewed by conventional transmission electron microscopy. MT, microtubules; R, polyribosomes; SF, actin stress fibers.
(From Heuzer JE, Kirschner M. J Cell Biol 1980;86:212, by permission of Rockefeller University Press.)

Figure 1–21 Cryoelectron microscopy and tomography of human immunodeficiency virus (HIV). Concentrated virus (HIV or SIV) in
aqueous suspension was placed on a grid and rapidly frozen by plunging the grid into liquid ethane at − 196°C. The frozen sample was
then placed in a cryoelectron microscopy grid holder for viewing at a magnification of × 43,200. The sample holder was tilted at a
succession of angles for consecutive images, from which tomograms were computed. (A) Sample virus field; the virus shown in this field
is simian immunodeficiency virus (SIV), which has a higher density of surface spike proteins than HIV. The virus particle indicated by the
arrow was chosen for tomographic analysis. (B) Computationally derived transverse sections through the selected virus particle (from top
to bottom). (C) Tomographic structure of the virus envelope spike complex, which is a trimeric structure of viral gp120 (globular portion of
the spike) and gp41 (transmembrane “foot”) proteins, in the form of a twisted tripod. (From Zhu P, et al. Nature 2006;441:847, by
permission.)

Figure 1–22 High-resolution scanning electron micrograph of nuclear pore complexes. (A) Purified nuclear envelopes were prepared for
scanning electron microscopy. The electron micrograph shows the image of nuclear pores as viewed from the nuclear side of the pore.
(B) Current model for the structure of a nuclear pore. ([A] From Goldberg MW, Allen TD. J Cell Biol 1992;119:1429, by permission of
Rockefeller University Press; [B] modified from Alberts B, et al. Molecular Biology of the Cell, 4th ed. New York, NY: Garland Science,
2002.)

Figure 1–23 Atomic force microscopy (AFM). In AFM the sample is scanned by a microscale probe, consisting of a sharp tip attached to
a flexible cantilever. The deflection of the probe as it moves over the sample is measured by the movement of a laser beam reflected from
the top of the cantilever onto an array of photodiodes. (Modified from the Wikipedia article “Atomic Force Microscope,”
http://en.Wikipedia.org/wiki/Atomic_force_microscopy.)

Figure 1–24 Atomic force microscopy (AFM) image of fusion pores in the membrane of a living cell. The apical plasma membrane of
living pancreatic acinar cells was scanned by AFM, producing this image of multiple pore structures. Pores are located in permanent pit
structures (one of which is framed by the white box) on the apical surface membrane. Inset: Schematic depiction of secretory vesicle
docking and fusion at a fusion pore. Fusion pores (blue arrows), 100–180 nm wide, are present in “pits” (yellow arrows). ZG, zymogen
granule. (From Hörber J, Miles M. Science 2003;302:1002, reprinted with permission from AAAS.)

Figure 1–25 Atomic force microscopy (AFM) “biopsy” of a human chromosome. Metaphase chromosome spreads were prepared and
fixed on glass microscope slides by standard techniques. Air-dried, dehydrated chromosomes were first scanned by AFM in noncontact
mode; for dissection (A) the probe was dragged through a previously identified location on a selected chromosome with a constant
applied downward force of 17 micronewtons. (B) Scanning electron microscopic image of the tip of the probe used for the dissection
shown in A; the material removed from the chromosome on the tip of the probe is circled. DNA in the sample could subsequently be
amplified by polymerase chain reaction. (From Fotiadis D, et al. Micron 2002;33:385 by permission of Elsevier Science, Ltd.)

Figure 1–26 Cell strain versus established cell lines. Murine cells (e.g., mouse embryo cells) initially grow well in culture, and during this
period of growth, such cells are termed a “cell strain.” But the growth rate falls after several generations, and the cells enter “crisis,”
following which almost all cells senesce and die. Often, however, a rare variant cell will arise in the culture, capable of indefinite growth
(i.e., “immortal”). The descendants of this variant cell become an “established cell line.” These immortalized cells are typically aneuploid.
(Modified from Todaro GJ, Green H. J Cell Biol 1963;17:299–313.)

Figure 1–27 Fluorescence-activated cell sorting (FACS). Antibodies tagged with red or green fluorescent molecules and specific each for
one of two different cell surface proteins (e.g., CD4 and CD8) are used to label a population of cells (e.g., a population containing the CD4
and/or the CD8 protein on their surface). The labeled cells pass into a vibrating flow cell, from which they emerge within individual fluid
droplets. The droplets are excited by a laser beam. Forward-scattered laser light, side-scattered laser light, and red and green fluorescent
light from the droplet are measured. Based on these measurements, individual droplets will be given a positive (+) or negative (−) charge
and then diverted to collection tubes via charged deflection plates. (Modified from Roitt IM, Brostoff J, Male D. Immunology, 5th ed. St.
Louis: Mosby Year-Book, 1998.)

Figure 1–28 Differential centrifugation. A cell lysate is placed in a centrifuge tube, which, in turn, is mounted in the rotor of a preparative
ultracentrifuge. Centrifugation at relatively low speed for a short time (800 g/10 min) will suffice to pellet unbroken cells and nuclei. The
supernatant of the low-speed spin is transferred to a new tube, and centrifuged at a greater speed and longer time (12,000 g/20 min) will
pellet organelles (mitochondria, lysosomes, and peroxisomes); centrifugation of that supernatant at high speed (50,000 g/2 h) will pellet
microsomes (small fragments of endoplasmic reticulum and Golgi membranes); centrifugation at very high speeds (300,000 g/3 h) will
pellet free ribosomes or viruses or other large macromolecular complexes. (Modified from Alberts B, et al. Molecular Biology of the Cell,
4th ed. New York, NY: Garland Science, 2002.)

Figure 1–29 Rate-zonal centrifugation versus equilibrium density gradient centrifugation. (A) In rate-zonal centrifugation the sample is
layered on top of a shallow sucrose gradient. During centrifugation the various components in the sample then move toward the bottom of
the tube based on their sedimentation coefficients. After resolution of the components, the bottom of the plastic tube is pierced and
fractions are collected. (B) Equilibrium density centrifugation resolves components in the sample based on their molecular density. The
sample is either layered onto or incorporated into a steep sucrose gradient; during centrifugation, individual components move in the
centrifugal field until they reach a density in the gradient that is identical to the buoyant density of the sample component. At this point,
each component stops moving and forms a band in the gradient. (Modified from Alberts B, et al. Molecular Biology of the Cell, 4th ed.
New York, NY: Garland Science, 2002.)

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