CHAPTER 3

Subcellular Fractionation and Isolation of

Organelles
INTRODUCTION
n the early 1700s, Leeuwenhoek noted that avian and amphibian blood cells contained
I a clear area, almost certainly corresponding to the structure we now know as the
nucleus. This represents probably the earliest recognition that eukaryotic cells are not
simply sacs of protoplasm, but instead contain subcellular structures. Although the con-
cept of the nucleated cell became firmly established in the 18th and 19th centuries, the
real beginning of subcellular fractionation had to await the emergence in the mid-1940s
of the art form known as electron microscopy. The electron microscope revealed much
more complexity than Leeuwenhoek could have imagined. Concurrent with improve-
ments in the techniques of electron microscopy was the development of methodologies
for subcellular fractionation. Through the 1950s, these parallel approaches resulted in
the discovery and isolation of the major organelles that comprise the eukaryotic cell. Fi-
nally, as biochemical functions were associated with specific subcellular compartments,
a much clearer picture of the eukaryotic cell began to emerge, and with it the field of
modern cell biology.

Chapter 3 is dedicated to methods for subcellular fractionation. The chapter begins with
an overview of cell fractionation (UNIT 3.1), which covers some basic principles as well
as the instrumentation of centrifugation. The various centrifugation media, ranging from
sucrose to the newer media (Ficoll, Percoll, metrizamide, Nycodenz, and Iodixanol), are
described.

Much of the earliest work in subcellular fractionation utilized rat liver as starting material.
The second unit of Chapter 3 provides protocols for the isolation of plasma membrane
sheets and domains from rat liver (UNIT 3.2). This method is used to isolate plasma
membrane sheets in sufficient yield and purity to be suitable for a variety of analytical
and preparative purposes. For example, these preparations can be used as starting material
for the isolation of hepatocyte plasma membrane proteins. The sheets also serve as the
starting material for the protocols within UNIT 3.2 that yield the apical and basolateral
domains comprising the polarized hepatocyte plasma membrane.

The densities of mitochondria, peroxisomes, and lysosomes are very similar, and as
a result, these organelles cosediment in density gradients as the so-called light mito-
chondrial fraction. The development of separation media has significantly improved the
ability to separate these organelles from each other. UNITS 3.3-3.6 deal with separation of
these organelles.

UNITS 3.3 & 3.4 describe methods for isolating mitochondria from rat liver as well as other
sources including other tissues, cultured mammalian cells, and yeast. UNIT 3.3 focuses on
methods involving differential centrifugation for rapid isolation of metabolically active
mitochondria, whereas methods involving density-gradient centrifugation for further
purification of mitochondria are described in UNIT 3.4.

UNIT 3.5provides methods for the isolation of peroxisomes from mammalian tissues Subcellular
and cells in tissue culture. Also included in this unit are a method for preparation of Fractionation
and Isolation of
Organelles

Current Protocols in Cell Biology 3.0.1-3.0.8, September 2011 3.0.1

Published online September 2011 in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/0471143030.cb0300s52 Supplement 52
Copyright C 2011 John Wiley & Sons, Inc.
 peroxisomes from yeast and descriptions of assays for assessing the success of subcellular
fractionation of the various starting materials.

The yeast Saccharomyces cerevisiae contains all the membrane-bound subcellular or-
ganelles that are characteristic of higher eukaryotes, yet this organism has a haploid
genome that is less than four times the size of that of E. coli. The availability of the entire
S. cerevisiae genome sequence, together with the ease with which yeast are cultured
(UNIT 1.6) and the simplicity with which genetic manipulations are performed have made
this organism a popular model in eukaryotic cell biology. Indeed, it is yeast cell biology
that may provide insights into the function of many yeast genes and their mammalian
counterparts. While some attention to certain subcellular organelles from yeast is given
in earlier units of this chapter, UNIT 3.7 provides a comprehensive overview of yeast
subcellular fractionation.

UNIT 3.8 describes centrifugation-based methods for isolating most of the major organelles
of the yeast Saccharomyces cerevisiae. Most of these procedures begin with the prepara-
tion of spheroplasts as described in the Support Protocol near the beginning of the unit.
Lysates for subcellular fractionation can also be generated by glass-bead lysis to disrupt
intact yeast cells (also described). Because of the fragility of most yeast organelles,
lysates of spheroplasts are best used for most protocols; however, glass-bead lysates
can be used for preparation of cytosol and plasma membranes. Several of the protocols
are analytical for assessing the distribution of proteins along exocytic, endocytic, and
biosynthetic pathways in yeast cells. The unit also includes preparative methods for iso-
lating yeast nuclei, vacuoles, mitochondria, peroxisomes, endoplasmic reticulum, plasma
membranes, and cytosol.

The Golgi, an organelle named for the pioneering cell biologist, Camillo Golgi, was
identified on the basis of a then current protocol developed by Golgi for fixing and
staining tissues. Although observations of what he then referred to as the internal
reticular apparatus probably date from even earlier in the 19th century, the Golgi remains
an intracellular site of intense investigation by todays cell biologists. UNIT 3.9 describes
the isolation of Golgi membranes on a preparative basis. Individual protocols describe
isolation of dextrose-treated Golgi stacks from rat liver using a sucrose density barrier
and by flotation from a light mitochondrial fraction. Isolation from cultured cells is
also described, as is a method to assay for the Golgi marker enzyme, UDP-galactose
galactosyl transferase.

UNIT 3.10 focuses on the clear area of Leeuwenhoekthe nucleus. As noted above, rat
liver has been the most frequently used source of material for subcellular fractionation.
This unit contains two protocols for isolation of nuclei from rat liver. The first utilizes
a sucrose density barrier, whereas the second employs Nycodenz as a density medium.
Although the vast majority of methods for preparing nuclei use soft tissue (e.g., liver),
the protocols described in this unit can be applied to other source materials (e.g., cul-
tured cells, plants), provided appropriate homogenization can be achieved. UNIT 3.10 also
contains two protocols for purification of nuclear membranes from isolated nuclei and
Support Protocols describing methods for assaying DNA and RNA.

Free-flow electrophoresis (UNIT 3.11) provides opportunities for both analytical and prepar-
ative applications for isolating subcellular organelles such as rat endosomes. Endosome-
enriched fractions are prepared from rat liver using discontinuous sucrose gradients,
and these are used to prepare endosomal subpopulations by free-flow electrophoresis.
The analytical approach can be used to study the kinetics and cellular subpopulations
involved in endocytic traffic. Three distinct subpopulations of endosomes are revealed in
such studies. Support Protocols include methods for analyzing markers for endosomal
Introduction subpopulations and for demonstrating retention of endosomal function (acidification)
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in vitro. Methods are also included for preparing and labeling ligands that traffic through
endosomes.

UNIT 3.12 describes protocols for the isolation of synaptic vesiclessmall, electron-lucent
organelles contained within presynaptic nerve terminals. Synaptic vesicles function to
store neurotransmitters and to release them into the synaptic cleft upon stimulation.
Methods for the purification of synaptic vesicles take advantage of the vesicles small
size and low density. The first two protocols describe the use of differential centrifu-
gation to enrich synaptic vesicles 8- to 10-fold relative to brain homogenate. The next
three protocols allow further purification to 20-fold using sedimentation velocity or
chromatography on controlled-pore glass beads or Sephacryl S-1000. This unit ends with
protocols for the sedimentation of synaptic vesicles at high speed, immunoisolation of
synaptic vesicle subpopulations, and siliconization of tubes.

Transport of proteins, lipids, and other cargo molecules between different compart-
ments of the secretory, endocytic, and lysosomal targeting pathways is mediated by
membrane-enclosed carriers. The best characterized of these carriers are clathrin-coated
vesicles (CCVs), which play critical roles in endocytosis from the plasma membrane and
sorting from the trans-Golgi network and endosomes. CCVs are small (50 to 100 nm
in diameter) and are encased within a dense proteinaceous coatproperties that are the
bases for their isolation by ultracentrifugation procedures. UNIT 3.13 presents a series of
protocols for the isolation of coated vesicles from different sources, including adult and
developing rat brain, adult rat liver, and cultured cell lines. In addition, this unit con-
tains a procedure for analyzing the purity of isolated CCVs by electron microscopy. The
Commentary section shows representative electron micrographs of these preparations,
as well as SDS-PAGE analyses of the protein composition of the purified CCVs.

UNIT 3.14 deals with melanosomes, which are membrane-bound organelles filled with
the pigment melanin. These organelles are only found in specialized cell types such as
melanocytes and retinal epithelial cells, and they function to protect cells and tissues from
the harmful effects of ultraviolet radiation. Oncogenic transformation of melanocyte
precursors results in melanomas, which are particularly aggressive cancers. This unit
presents protocols for the isolation of melanosomes from melanoma cells and tissues.
The biogenesis of melanosomes involves maturation of precursors through a series of
well-defined stages; the protocols in this unit allow for the separation of early and late
stage melanosomes by ultracentrifugation and free-flow electrophoresis. This unit also
describes a method for monitoring the purification of melanosomes by measuring the
activity of the melanosomal enzyme tyrosinase. Finally, the unit lists antibody reagents
to melanosomal proteins that can be used to monitor purification and assess the purity of
the isolated melanosomes.

Most cells in culture contain discernable lipid droplets, although the size and number
of these droplets vary depending on cell type and culture conditions. Lipid droplets can
provide a source of metabolic energy and also play a role in cholesterol homeostasis
required for membrane biogenesis. In steroidogenic cells, the lipid droplets provide
substrates for hormone synthesis. UNIT 3.15 describes the isolation of lipid droplets based
on their high buoyancy, which is reflective of their low mass of protein and high relative
mass of neutral lipids. At present, no specific enzyme markers have been localized
exclusively to lipid droplets. The droplets do, however, contain certain characteristic
structural proteins, and protocols for immunoblotting of these proteins are included for
assessment of the isolated lipid droplets.
Subcellular
UNIT 3.16describes the isolation of rat serosal mast cells by peritoneal lavage and the use Fractionation
of these cells as a source of mast cell granules. These granules are modified lysosomes and Isolation of
that release their contents upon degranulation of the activated mast cells. Protocols Organelles

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 are provided for the isolation of mast cell granules enveloped in their perigranular
membrane and of granule remnants released upon degranulation. Several protocols aimed
at assessing mast cell activation are included, as is a protocol for verification of granule
remnant morphology and purity via electron microscopy. A future unit in Chapter 3 will
deal with the isolation of the analogous organelle from chromaffin cells (i.e., chromaffin
granules).

All previous units in this chapter that deal with organelle isolation have described isolation
of organelles that exist in multiple copies per cell. In contrast, UNIT 3.17 provides protocols
related to the isolation of centrosomes, of which there is only one per cell. As starting
material for this challenging task, the protocols utilize early (0- to 3.5-hr) Drosophila
embryos. Centrosomes are isolated by a protocol involving gradient centrifugation and a
protocol for further enrichment via immunomagnetic purification. Protocols for assessing
the centrosome preparation by immunofluorescence microscopy are included.

In contrast to the situation with the centrosome (one copy per cell), pancreatic zymogen
granules have been estimated to constitute as much as 25% of the total cellular volume
based on electron microscopy. The relative abundance of these organelles resulted in
their being among the first to be the subject of cellular subfractionation protocols, dating
to the mid-1950s. UNIT 3.18 provides more modern protocols for the purification of
these important cellular bodies from both rat and dog pancreas, using both sucrose and
Percoll gradients. The pancreas contains other secretory granules in lower abundance,
most notably the endocrine granules responsible for insulin release. A future unit of this
chapter will provide protocols related to isolation of the insulin granules of the pancreas.

An early unit in this chapter (UNIT 3.5) deals with the organelles known as peroxisomes.
Glyoxysomes represent a specialized form of the peroxisome found in the cotyledons of
seedling plants. In this context, these organelles sequester the enzymes of the glyoxylate
cycle that provides energy to the rapidly growing seedling. Later in plant development, the
peroxisomes of the green leaves have a distinct complement of enzymes reflecting their
role in photorespiration. UNIT 3.19 provides protocols for growing pumpkin seedlings and
for isolation of glyoxysomes from their cotyledons via density gradient centrifugation.

The glucose transporter isoform known as GLUT4 functions to facilitate uptake of glu-
cose by both fat and muscle tisssues. GLUT4 is translocated in, response to insulin, from
an intracellular compartment to the plasma membrane, as the mechanism for insulin-
mediated enhancement of glucose transport. The investigation of trafficking of GLUT4
has gained considerable attention since the discovery that GLUT4 translocation is com-
promised in type 2 diabetes. UNIT 3.20 provides protocols for isolation of GLUT4 storage
vesicles from primary rat adipocytes, cultured adipocytes, and skeletal muscle.

Another type of membrane vesicle is the subject of UNIT 3.21, which describes isolation
of vesicles from intestinal brush border from three species (rat, pig, and cow). These
vesicles are derived from the apical surface of the polarized epithelial cells of the small
intestine, which also contain integral membrane proteins involved in glucose uptake.
Whereas the GLUT4 vesicles of UNIT 3.20 are part of a regulated trafficking of cellular
membranes, those of UNIT 3.21 represent spontaneous sealing of membrane fragments
derived from the intestinal microvilli upon disruption of the tissue architecture. These
vesicles can nonetheless be utilized for membrane transport studies.

The theme of isolation of membrane-derived vesicles continues in UNIT 3.22, which pro-
vides several protocols related to exosomes. These vesicles are derived from the plasma
membrane via an endocytosis-exocytosis mechanism, but they are isolated from extracel-
lular fluids (either tissue culture media or bodily fluids). Methods are provided whereby
Introduction exosomes are isolated by either ultracentrifugation, ultrafiltration, or immunoisolation.
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The protocols of UNIT 3.22 should prove useful in studies aimed at understanding ex-
osome function and the possible use of exosomes in therapeutic applications e.g., as
immunogens in vaccines.

UNIT 3.23 describes the isolation of intermediate filaments from bovine spinal cord. Al-
though intermediate filaments from different sources contain a diverse complement of
intermediate filament proteins, all intermediate filaments share the property of being
insoluble in conventional extraction buffers. It is this property that serves as the basis
of the isolation protocol. Intermediate filaments can also be produced after assembly of
a recombinant intermediate filament protein, and an Alternate Protocol describes this
method. With intermediate filaments isolated from either tissue or assembled recom-
binant protein, it is possible to study the assembly process in vitro and to investigate,
in vitro, possible interaction partners that participate in intermediate filamentmediated
processes in vivo.

Skeletal and cardiac muscle cells contain an unusual system of tubular invaginations of
their sarcolemma known as the transverse tubule (T-tubule), which are critical participants
in the process of muscle contraction. UNIT 3.24 provides a Basic Protocol that enables
the isolation of highly purified T-tubules from muscle tissue using density gradient
ultracentrifugation. A second, related Basic Protocol takes advantage of the glycoproteins
in the membranes to separate sarcolemma membrane vesicles from T-tubule vesicles via
aggregation of the former by wheat germ agglutinin. An Alternate Protocol involves
immunoisolation of T-tubule vesicles utilizing antibodies that recognize the intracellular
domain of T-tubule integral membrane proteins (e.g., GLUT4).

Another very specialized membrane is the myelin sheath that surrounds vertebrate axons
in both the central and peripheral nervous system. CNS myelin is elaborated as a plasma
membrane extension by oligodendroglial cells of the brain and spinal cord, whereas PNS
myelin is generated by Schwann cells. Methods for isolating myelin go back over three
decades, and the methods of UNIT 3.25 are in fact based on the classical isolation method
of Norton and Podusulo published in 1973. The methods here are modified to minimize
proteolysis and contamination with the axon plasma membrane. Although the protocols
are related, separate protocols are provided for isolation of myelin from the CNS and
from the PNS, reflecting the morphological and biochemical distinction in the two forms
of myelin. In both cases, the relatively low density of myelin-derived membrane vesicles
allows for a high degree of purification. A support protocol for assay of a marker enzyme
is also included.

Brush borders also represent a specialization of the cell surface. In this case, rather than
wrapping around axons in a spiral fashion as does myelin, the brush border is charac-
terized by characteristic folds of the plasma membrane to form finger-like projections
termed microvilli. These structures markedly increase the surface area of the membrane,
which enables enhancement of absorption of substances. UNIT 3.26 provides protocols for
the isolation of brush borders from the kidney where the brush border structure parti-
cipates in glomerular filtration. The microvilli that make up the renal brush border are
closely associated with actin filaments, explaining why brush borders have been utilized
as a source of actin-binding proteins. It also accounts for the fact that brush borders
are relatively resistant to homogenization, and this feature is exploited in the isolation
protocols. Isolated brush borders can be further studied and purity/integrity assessed by
flow cytometry, marker enzymes, and/or electron microscopy.

The boundary between the fields of virology and cell biology has always been blurry
Subcellular
due to the fact that viruses infect cells. The cellular pathways of protein trafficking are Fractionation
among those that are hijacked by many viruses. Indeed, the trafficking of viral proteins and Isolation of
has been instrumental in understanding these pathways. To localize and examine the Organelles

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 trafficking of the proteins of human cytomegalovirus (HCMV), the ability to isolate
subcellular fractions from virally infected cells has proven to be highly informative.
UNIT 3.27 provides procedures for isolating mitochondria, endoplasmic reticulum, and
mitochondria-associated membranes from primary fibroblasts and HeLa cells infected
with HCMV. The unit also includes a protocol for visualization of the disruption of the
mitochondrial network based on subcellular fractionation in Percoll gradients.

Amyloplasts are organelles wherein starch is synthesized and stored in plants. The
starch in amyloplasts can subsequently be converted to sugar to meet the energy needs
of the plant. Specialized amyloplasts are also thought to participate in gravitropism
(sensing of gravity) that determines the growth of root tips. Much less is known about
the biochemistry and function of these organelles than other, more studied plastids (e.g.,
chloroplasts). In UNIT 3.28, protocols are provided for the isolation of amyloplasts from
maize endosperm and oilseed rape embryos as starting material. These methods could
be adapted for other sources.

Microtubules are one of the components of the cytoskeleton. They are dynamic structures
undergoing an assembly-disassembly process that contribute to cell motility, movement
of organelles within the cell, and segregation of chromosomes into the daughter cells
during mitosis. Microtubules are polymers of -tubulin and -tubulin with associated
proteins (the microtubule-associated proteins or MAPs). The - heterodimer is the
basic building block of microtubules, and a classical method for the isolation of the
soluble heterodimer is provided in UNIT 3.29. Also described in this unit are protocols
aimed at analysis of tubulin and MAPs. These protocols include the chromatographic
and electrophoretic methods that have been traditionally employed, but they also include
a new protocol for visualizing microtubules by atomic force microscopy.

Perhaps the best known organelle in plants is the chloroplast, which is responsible
not only for energy production via photosynthesis but also for other critical metabolic
functions, including synthesis of amino acids, vitamins, and lipids. UNIT 3.30 provides
protocols for the purification of chloroplasts from both Arabidopsis and spinach leaves.
The purification is accomplished using differential centrifugation followed by Percoll
gradient centrifugation and yields intact organelles suitable for additional characterization
and experimentation. Whereas chloroplasts have been classically isolated from spinach
leaves, the inclusion of an isolation protocol tailored for using Arabidopsis leaves as
starting material is of significance, owing to the widespread use of this organism in plant
genetics and molecular biology.

Melanins are pigmented polymers that occur widely in both plants and animals. Cu-
taneous melanin is responsible for skin color. Melanin in the brain has been termed
neuromelanin and is located in the human midbrain region known as the substantia
nigra pars compacta. Indeed the name of this portion of the brain is related to its pig-
mented nature (from the Latin meaning black body). This region of the brain is related
to control of movement, and loss of the pigmentation in this region is a feature of
Parkinsons disease. Neuronal neuromelanin granules are distinct from melanosomes
(see UNIT 3.14), which are located in melanocytes and are thought to be related to lyso-
somes. The neuromelanin granules exhibit a wider range of size than do melanosomes
and have a distinct lipid composition. UNIT 3.31 describes the isolation of neuromelanin
granules with human midbrain as the starting material.

Dense core secretory vesicles (also termed secretory granules) are present in endocrine
and neuroendocrine cells where they serve as storage sites for hormones (e.g., insulin),
proteases, and signaling molecules. It appears that the intracellular sorting of peptide
hormone precursors to the dense core secretory vesicles is an essential step in the bioac-
Introduction tivation of these precursors. UNIT 3.32 details the isolation of dense core vesicles from
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pancreatic exocrine cells utilizing differential and density-gradient centrifugation. The
unit describes isolation of the vesicles from pancreatic islets, as well as from tissue
culture lines and transplantable tumors.

Alzheimers disease is characterized by lesions in the brain that contain deposits of

amyloid- protein (plaques) as well as neurofibrillary tangles consisting of abnormally
phosphorylated microtubule-associated tau protein. A fuller understanding of these struc-
tures will help elucidate the pathology of the disease. UNIT 3.33 provides protocols for the
isolation, biochemical analysis, and characterization of both amyloid plaques and neu-
rofibrilar tangles.

Legionella pneumophila is the bacterial pathogen responsible for the disease known as
Legionnaires disease. The name is based on an outbreak of deadly pneumonia at a
convention of the American Legion in Philadelphia in 1976. The previously unknown
bacterium was isolated the following year. This organism replicates intracellularly within
phagocytes in a unique compartment that is called the Legionella-containing vacuole
(LCV). It is now appreciated that the LCV is formed by hijacking the host signaling and
vesicle trafficking pathways and involves a large number of bacterial effector proteins.
The method of isolation of LCVs described in UNIT 3.34 utilizes immunomagnetic sepa-
ration using an antibody to one of the bacterial proteins on the surface of the vacuoles,
followed by density gradient centrifugation. The unit also provides a means of monitoring
the enrichment of the LCVs via fluorescence microscopy.

Immunomagnetic methods are also employed in one of the protocols of UNIT 3.35 for the
isolation of a distinct intracellular structure, the platelet granule. Platelets possess alpha
granules that contain several growth factors, as well as dense (delta) granules. An alterna-
tive protocol utilizes more traditional differential centrifugation/sedimentation method-
ology. The degree of purification is monitored via immunoblotting using antibodies that
recognize specific granule proteins.

Earlier in this chapter, there is a unit which is focused on isolation of nuclei from rat liver
(see UNIT 3.10). Within nuclei, the nucleoli are non-membrane-bound structures composed
of RNA and protein. These domains within the nucleus are involved in a number of
important cellular functions, notably ribosome biogenesis and tRNA maturation. The
purification of these relatively large and dense structures from the nuclei of cultured cells
is described in UNIT 3.36.

Killer lymphocytes have been known for about 50 years, following their discovery in
the context of transplant rejection in vivo. Two of the varieties of killer lymphocytes
are cytotoxic T cells and natural killer cells. Each of these cell types contains cytotoxic
granules, the contents of which are responsible for elimination of target cells. The
protocols of UNIT 3.37 describe purification of such granules utilizing Percoll density
gradients following disruption of the killer lymphocytes. The unit also contains protocols
for purification via liquid chromatography of key proteins involved in the cytolytic
process.

Proteinaceous inclusion bodies formed when the cellular proteosome degradation mech-
anism is impaired are known as aggresomes. These structures form in the vicinity of the
microtubule organizing center and centrosome and appear to be associated with inter-
mediate filaments. The aggresomal response is thought to be a protective mechanism for
dealing with abnormally high cytosolic loads of abnormal (e.g., misfolded) or damaged
protein. Neuronal cytosolic inclusion bodies seen in Parkinsons disease are thought to
be aggresomes. UNIT 3.38 provides a protocol for isolating aggresomes from a yeast model. Subcellular
Fractionation
and Isolation of
Organelles

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 Chromaffin cells are neuroendocrine cells found in the medulla of the adrenal gland.
These cells contain secretory vesicles (granules) that contain a complex mixture of cate-
cholamine neurotransmitters, peptide hormones, and a number of enzymes. Understand-
ing of the molecular composition of chromaffin granules continues to evolve. The two
protocols of UNIT 3.39 should further this understanding, as relatively large amounts of the
granules can be obtained in a relatively short time. One protocol utilizes an iso-osmotic
medium and differential centrifugation and yields intact granules. The second is used for
isolation of chromaffin granule membranes or contents for further characterization.

The cellular structure at the heart of the process of protein synthesis is the ribosome.
These electron-dense 10- to 15-nm spheres in the cytoplasm of cells were first ob-
served via transmission electron microscopy by George Palade in 1955. Subsequently,
their structural features as ribonucleoprotein complexes with three RNA molecules and
over forty proteins have come to be understood more clearly. Indeed, the three dimen-

sional structure of the 70S bacterial ribosome has been elucidated at 5.5-A resolution by
X-ray crystallography. Eukaryotic ribosome structure has been probed by cryo-electron
microscopy. There is evidence that cells dedicate a significant fraction of their energy to
maintenance of their ribosomal content, which is higher in proliferative than quiescent
cells. UNIT 3.40 provides a Basic Protocol for the purification of ribosomes from cultured
human cell lines that is adaptable to other animal cell lines. A Support Protocol for SDS-
polyacrylamide gel electrophoresis to assess purity of the ribosomal fraction obtained.
While other protocols for ribosome purification exist, the protocol of UNIT 3.40 has been
demonstrated to be robust and has been optimized and validated for cultured human cell
lines.

Joe B. Harford and

Juan S. Bonifacino

Introduction

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