An Overview of Methods in

All present-day cells are descended from

Cell Biology a single primordial ancestor.
The first cells emerged at least 3.8 billion
years ago.

Cells as experimental models

Laboratory Methods
Because the fundamental properties of
all cells have been conserved during
evolution, the basic principles learned
and
from experiments on one type of cell are
generally applicable to other cells. Experimental Tools
Tools of Cell Biology The cellular structure of cork

Many important advances in cell biology research

have directly followed the development of new
methods that have opened novel avenues of
investigation.
The discovery of cells arose from the development of
the light microscope.
Robert Hooke coined the term “cell” following his
observations of a piece of cork in 1665.
In the 1670s Antony van Leeuwenhoek was able to
observe a variety of cells, including sperm, red blood
cells, and bacteria.

Cell theory Tools of Cell Biology - Microscope

The cell theory proposed by Matthias Schleiden and
Theodor Schwann in 1838 resulted from their
studies of plant and animal cells using microscopes.
It was soon recognized that cells are not formed de
novo but arise only from division of pre-existing cells.
Contemporary light microscopes can magnify
objects up to about 1,000 x.
Most cells are between 1–100 µm, so they can
be observed by light microscopy, as can some
organelles.
Resolution: the ability to distinguish objects
separated by small distances; is even more
important than magnification.
Light microscope resolution

The limit of resolution of the light microscope

is approximately 0.2 µm. Objects separated by
less than that distance appear as one object.
This limit is determined by the wavelength of
visible light (λ), and the numerical aperture
(NA): the light-gathering power of the lens.

Numerical Aperture
N.A. = n x (sin µ)
0.61l
Resolution =
NA
λ is fixed at approximately 0.5 mm
NA can be envisioned as the size of the cone
of light that enters the lens.

NA = h sin a
η = refractive index of the medium.
 η = 1.0 for air, but can be increased to a
maximum of 1.4 by using an oil-immersion
lens.
Maximum for α is 90 , at which sin α = 1, so
maximum possible for NA = 1.4.
The theoretical limit of resolution is thus:

0.61 ´ 0.5
Resolution = = 0.22 µm
1.4

Types of light microscopy 1 Types of light microscopy 2

Bright-field microscopy: light passes directly
through the cell. Cells are often preserved with
fixatives and stained with dyes to enhance the
contrast.
Phase-contrast microscopy and Differential
interference-contrast microscopy (DIC or
Normarski) both convert variations in density or
thickness to differences in contrast that can be seen
in the final image.
 Types of light microscopy 3
Video cameras and computers for image analysis
and processing can substantially enhance the
contrast of images.
Video-enhanced differential interference-
contrast microscopy allows visualization of the
movement of organelles along microtubules,
cytoskeletal protein filaments with a diameter of only
0.025 µm.

Types of light microscopy 4

Fluorescence microscopy – A fluorescent dye is

used to label the molecule of interest in fixed or
living cells.
This fluorescence is detected by illuminating the
specimen with a wavelength of light that excites the
fluorescent dye, then using filters to detect the
specific wavelength of light that the dye emits.

505DCLP 480/30x 535/40m

Dichroic Mirror Exciter Emitter
GFP is a powerful reagent for live cells
Fluorescence recovery after photobleaching
fluorescent microscopy (FRAP) is used to study rate of protein movement in
living cells.
The green fluorescent protein (GFP) of jellyfish can
be used to visualize proteins in living cells.
GFP can be fused to any protein of interest using
standard methods of recombinant DNA.
 Fluorescence resonance energy transfer (FRET)
is used to study interactions between proteins in a
cell.
Types of light microscopy 5
The images of conventional fluorescence microscopy
can be improved by image deconvolution – a
computer analyzes images obtained from different
depths of focus and generates a sharper image from
them.

Types of light microscopy 6

Confocal microscopy
increases contrast and
detail by analyzing
fluorescence from a single
point.
 The emitted light must pass through a pin-hole
aperture (confocal aperture). Thus only light emitted
from the plane of focus is able to reach the detector.
Scanning across the specimen generates a two-
dimensional image of the plane of focus.
Types of light microscopy 8
Super-Resolution Fluorescence Microscope
STORM (stochastic optical reconstruction
microscopy) allows the localization of a single
fluorescent molecule within a resolution of <20 nm.
Types of light microscopy 7
Multi-photon excitation microscopy
The localization of excitation minimizes damage to
the specimen, allowing three-dimensional imaging of
living cells.
Electron microscopy
Electron microscopy can achieve much greater
resolution (0.2 nm) than light microscopy because
of the short wavelength of electrons.
Resolution for biological samples is about 1 to 2
nm because of their inherent lack of contrast.
Transmission electron microscopy
Specimens are fixed and stained with salts of heavy
metals, which provide contrast by scattering
electrons.
A beam of electrons is passed through the specimen
and forms an image on a fluorescent screen.

Negative staining methods for TEM observation

 Shadow Casting is a technique used to Electron tomography
visualize the surface of subcellular structures or generates three-
macromolecules. dimensional images by
The specimen is sprayed with a thin layer of computer analysis of
metal, such as platinum, from an angle which multiple two-dimensional
results in a shadowing effect. images obtained over a
range of viewing directions.

Freeze-fracture replication
and etching
This often splits the lipid
bilayer, revealing the interior
faces of a cell membrane.

Deep Etching
Scanning electron microscopy Scanning Electron Atomic Force Microscopy
provides a three-dimensional image of cells
Atomic Force
The surface of the cell is coated with a heavy metal, Microscopy (AFM) is
and a beam of electrons is used to scan across the a high-resolution
specimen. scanning instrument
that provides an
image of each
individual molecule
as it is oriented in
the field.

The Use of Radioisotopes Autoradiography

Biochemical methods • Autoradiography is a

technique to where a
particular isotope is
located.
§ A particle emitted from Preparation of a
a radioactive atom light microscopic
activates a photographic autoradiograph.
emulsion.
§ The location of the
radioisotope in the
specimen is determined
by the positions of the
overlying silver grains in
a photographic
emulsion.
Cell Culture
• Most of the study of cells is carried out using cell culture.
§ Cells can be obtained in large quantities.
§ Most culture contain a single type of cell.
§ Many different types of cells can be grown in culture.
§ Cell differentiation can be studied in a cell culture.
Embryos or tumors are frequently used as
starting material because they contain rapidly
growing cells and can proliferate indefinitely to
become immortal cell lines.
Embryo fibroblasts grow particularly well in
culture and are one of the most widely studied
types of animal cells.
Embryonic stem cells can be established in
culture and maintain their ability to
differentiate into all of the cell types of adult
organisms.
An initial cell culture
from tissue is a primary
culture.
Most normal cells such
as fibroblasts cannot be
grown in culture
indefinitely.
The culture media for animal cells are complex and
must include salts and glucose, and various amino
acids and vitamins that cells can’t make for
themselves.
Serum provides polypeptide growth factors. The
identification of individual growth factors makes it
possible to culture cells in serum-free media.
Harry Eagle was the first researcher to describe a
defined medium for animal cells, in 1955.
Plant cells can also be cultured. Cell Culture 2D vs 3D
Given appropriate growth factors, plant cells
produce a mass of undifferentiated cells called a Comparison of the
callus. morphology of cells
growing in 2D versus 3D
Many plant cells are capable of differentiation into cultures
many different cell types and even an entire plant.

• A two-dimensional culture system is when cells are grown on the flat

surface of a dish.
• Labs are moving to three-dimensional cultures in which cells are grown in
a 3D matrix consisting of extracellular materials.
• 3D cultures are better suited to study cell-cell interactions.

Subcellular fractionation

In order to determine the function of organelles,

they must be isolated from the cell.
Differential centrifugation separates cell
components on the basis of size and density.
The plasma membrane and ER is broken into small
fragments by sonication, grinding, or high-speed
blending.
The suspension is fractionated in an
ultracentrifuge.
Larger, more dense organelles sediment at lower
speeds; small organelles at high speeds.
Figure 1.38 Subcellular fractionation (Part 2)
Density-gradient centrifugation
In velocity centrifugation, particles of different
sizes sediment through the gradient at different
rates.
Figure 1.38 Subcellular fractionation (Part 3)
Equilibrium centrifugation in density gradients is
used to separate subcellular components on the
basis of their buoyant density.
The sample particles are centrifuged until they
reach an equilibrium position at which their buoyant
density is equal to that of the surrounding sucrose
or cesium chloride solution.
Isolation, Purification, and Fractionation of Proteins Isolation, Purification, and Fractionation of
Proteins Liquid column chromatography
• Protein purification involves the stepwise removal • Liquid Column Chromatography
of contaminants. § Chromatography includes a variety of techniques in
§ Purification is measured as an increase in which a mixture of dissolved components is
specific activity of a protein. fractionated through a porous matrix.
§ Some identifiable feature of the specific • Components are fractionated between mobile
protein must be utilized as an assay to and immobile phases.
determine the relative amount of the protein. • The greater the molecule’s affinity for the matrix,
the slower its movement.
• High performance liquid chromatography (HPLC)
has greater resolution due to a tightly packed
matrix.

Isolation, Purification, and Fractionation of Isolation, Purification, and Fractionation of

Proteins Gel filtration chromatography Proteins Ion-exchange chromatography

• Gel filtration separate • Ion-exchange

proteins by molecular weight. chromatography uses ionic
charge as a basis for
§ A column is packed with
purification.
cross-linked
polysaccharides of § DEAE-cellulose is positively
different porosity. charged and therefore binds
§ Proteins small enough to negatively charged
enter the pores are eluted molecules; it is an anion
last. exchanger.
§ Larger proteins unable to § CM-cellulose is negatively
enter the beads remain charged and acts as a cation
dissolved in the moving exchanger.
solvent phase.
Isolation, Purification, and Fractionation of Isolation, Purification, and Fractionation of
Proteins Affinity chromatography Proteins Polyacrylamide Gel Electrophoresis
• Affinity chromatography
• Polyacrylamide Gel
isolates one protein from a
Electrophoresis
mixture using a specific
§ Electrophoresis is based on
ligand.
the migration of proteins in
§ Proteins interact with an electric field.
specific substances: e.g. • In polyacrylamide gel
enzymes with substrates, electrophoresis (PAGE),
receptors with ligands, proteins are driven
antigens with antibodies. through a gel matrix.
• Movement of proteins
depends on molecular size,
shape, and charge density.

Isolation, Purification, and Fractionation of Isolation, Purification, and Fractionation of

Proteins SDS-Polyacrylamide Gel Electrophoresis Proteins 2D Gel Electrophoresis

• Polyacrylamide Gel • Two-Dimensional Gel

Electrophoresis Electrophoresis
§ SDS-PAGE § It separates proteins on
• Sodium dodecyl sulfate the basis of both
(SDS). isoelectric focusing and
• The repulsion between molecular weight.
bound SDS molecules •The technique is ideal
causes the proteins to for detecting changes in
unfold into a similar the proteins in a cell
shape. under different
• Proteins become conditions.
separated solely on the
basis of mass.
Two-dimensional gel electrophoresis Isolation, Purification, and Fractionation of
Proteins Spectrometry
Proteins are separated based on charge and size.
• Protein Measurement and Analysis
This technique is biased toward the most abundant
§ The amount of protein can be
proteins.
determined measuring the
amount if light absorbed using a
spectrophotometer.
§ Mass spectrometry (MS)
measures the mass of
molecules, determines chemical
formulas and molecular
structure, and identifies
unknown substances.

Mass spectrometry Determining the Structure of Proteins and Multisubunit Complexes

• X-ray crystallography (or X-ray diffraction) uses protein crystals.

§ Crystals are hit with X-rays, and scattered radiation is collected on a
photographic plate.
§ The diffraction pattern provides information about the structure of a protein.
§ The technique is useful in the study of both proteins and nucleic acids.
Determining the Structure of Proteins and Multisubunit Complexes Determining the Structure of Proteins and
Multisubunit Complexes

• Information gathered for a protein • Alternate techniques:

depends on the resolution
§ In myoglobin, 6 Å resolution shows how placed on a grid and rapidly frozen in a
polypeptide chain is folded and the hydrated state in liquid nitrogen without
location of the heme moiety.
§ At 2 Å resolution, groups of atoms can be
separated from one another.
§ At 1.4 Å, the positions of individual atoms
can be determined.
§ To date, the structures of several hundred
proteins have been determined at atomic
resolution (1.2 Å) and a few as low as 0.66
Å.
§ In electron cryomicroscopy particles are
being fixed or stained.
§ X-ray crystallography and electron
microscopic reconstructions to show
how the individual molecules that make
up a multisubunit complex interact.
§ Electron crystallography analysis of
frozen specimens can also be utilized in
the study of membrane proteins in the
lipid bilayer.
Combining data from electron
microscopy and X-ray
crystallography.

Proteomics: large-scale analysis of cell proteins

The proteome is all of the proteins expressed in a

given cell. A “shot-gun” approach eliminates the gel
The systematic analysis of cell proteins (proteomics)
electrophoresis.
aims to identify all proteins expressed in a cell, Cell proteins are digested with protease
where they are expressed, and their interactions.
and the whole mixture sequenced by
The number of genes expressed in any given cell is tandem mass spectrometry.
thought to be around 10,000.
Because of alternative splicing and protein
modifications, it is estimated that these genes give
rise to more than 100,000 different proteins.
Proteomics goals also include determining the The systematic analysis of protein complexes and
locations of proteins in cells and organelles. interactions has become an important goal of
proteomics.
Organelles are isolated by subcellular fractionation.
The proteins can then be analyzed by mass
spectrometry. • Proteins can be isolated
as complexes and
The subcellular analyzed by mass
localization of the spectrometry.
GFP-tagged protein
could then be • Alternative approaches
determined by like the yeast two-hybrid
fluorescence analysis are also used.
microscopy.

Recombinant DNA technology

Restriction endonucleases

Gel electrophoresis

Restriction maps
Restriction fragment length polymorphism
 Recombinant DNA Technology staggered sites – overhanging single-stranded tails
complementary base pairing

• DNA cloning is a
technique to produce
large quantities of a
specific DNA segment.
§ DNA segment to be
cloned is first linked to a
vector DNA.
§ Bacterial plasmids and
viruses are two
commonly used vectors.

cDNA cloning

reverse transcriptase

DNA ligase
cDNA
Cloning in plasmid vectors
Plasmids have an origin of replication and carry
antibiotic resistant genes.

Viruses provide simple systems that can be used to

investigate the functions of cells.
Bacterial viruses (bacteriophages) have simplified
the study of bacterial genetics.

plaques
 For even larger fragments of DNA, 5 major
Bacteriophage λ vectors can accommodate larger types of vectors are used.
fragments of insert DNA.
Sequences that aren’t needed for virus replication
are removed and replaced with unique restriction
sites for insertion of cloned DNA.
The recombinant molecules are then put into E.
coli, where they replicate to yield millions of
progeny phages containing a single DNA insert.

1. Cosmid vectors contain bacteriophage λ

sequences for packaging, origins of
replication, and genes for antibiotic
resistance, so they are able to replicate as
plasmids in bacterial cells.
2. Bacteriophage P1 vectors allow
recombinant molecules to be packaged in
vitro into P1 phage particles and be
replicated as plasmids in E. coli.
3. P1 artificial chromosome (PAC) vectors
also contain sequences of bacteriophage
P1 but are introduced directly as plasmids
into E. coli.
4. Bacterial artificial chromosome (BAC)
vectors are derived from a naturally
occurring plasmid of E. coli (the F factor).
5. Yeast artificial chromosome (YAC)
vectors contain yeast origins of replication
and other sequences that allow them to
replicate as linear chromosome-like
molecules in yeast cells.
Recombinant protein expression
expression vector
High levels of gene expression in bacteria or
eukaryotic cells
expression vector

Expression in eukaryotic cells may be needed

if posttranslational modification is required.
Cloned genes are inserted in virus vectors that
direct high-level gene expression.
One system uses infection of insect cells by
baculovirus vectors.
Polymerase chain reaction (PCR) Heat-stable DNA polymerases from bacteria, such as
Developed by Kary Mullis in 1988 for isolating large Thermus aquaticus
amounts of a single DNA molecule

DNA sequencing

Premature termination of DNA synthesis with

dideoxynucleotides
DNA Sequencing Newer techniques Nucleic Acid Hybridization

• Next-generation sequencing (NGS) is based on polymerase-

dependent DNA synthesis but does not depend upon
premature termination. Determining the
§ NGS does not require separation of strands by electrophoresis.
location of
§ It identifies the individual nucleotides as they are being incorporated
specific DNA
by the polymerase in real time. fragments in a
gel by a
• Third-generation (or single molecule) sequencing Southern blot
§ Does not require amplified DNA, nor do they involve enzymatic
activities. • Nucleic acid hybridization is based on the ability of two complementary DNA
strands to form a double-stranded hybrid.
§ Accomplishes sequencing by pulling each DNA molecule through a
tiny hole, or nanopore and identifying each nucleotide one at a • The Southern blot technique is based upon DNA hybridization.
time. • The Northern blot technique is based upon RNA-DNA hybridization.
• Hybridization can be used to determine the degree of similarity between two
samples.
Detection of DNA by nucleic acid hybridization
Southern blotting
vs.
Northern blotting
A DNA microarray is a glass slide or membrane filter Fluorescence in situ hybridization (FISH)
onto which oligonucleotides or fragments of cDNAs
are printed by a robotic system.

Detection of proteins
Antibodies can be used as protein probes.
Three common methods for using antibodies:
1. Immunoprecipitation
 2. Immunoblotting (Western blotting) 3. Immunofluorescence microscopy

The Use of Antibodies Genetic manipulation

Temperature-sensitive mutants
• A monoclonal antibody is
produced by descendants
• permissive temperature
of a single antibody- • nonpermissive temperature
producing cell:
§ Fusion of a normal
antibody-producing
lymphocyte and a
malignant myeloma cell
will create a viable
hybridoma cell that can
produce large amounts
of a monoclonal
antibody.
 Gene transfer – transfection
microinjection, coprecipitation, liposomes, electroporation

In most of the cells, the DNA is transported to The retroviruses have RNA genomes but
the nucleus, and is transcribed for several synthesize a DNA copy of their genome in infected
days – transient transfection. cells.

In 1 % or less of the cells, the foreign DNA is

integrated into the genome and is transferred
to progeny cells at cell division – stable
clones.
Retroviral transfection

Transgenic mice – pronucleus microinjection Gene knockout by homologous recombination –

occurs frequently in yeast but is rare in mammalian
cells.
Gene knockout mice
Knockout mice –
embryonic stem (ES)
cells transfection

For plant cells, DNA-coated microprojectiles,

such as small particles of tungsten, can be
shot directly into the plant cells by gene guns.
In many plants, a plasmid from the bacterium
Agrobacterium tumefaciens (Ti plasmid) is
used to introduce cloned DNA.
In nature, Agrobacterium attaches to the
leaves of plants and the Ti plasmid is
transferred into plant cells where it becomes
incorporated into chromosomal DNA.

Other approaches are used to interfere with gene
expression or function – the antisense nucleic
acids inhibition.
In vitro mutagenesis – Sometimes called reverse
genetics, since a mutation is introduced into a
gene first and its functional consequence is
determined second.
RNA interference (RNAi) was first discovered in C.
elegans in 1998, when Fire and Mello found that
injection of double-stranded RNA inhibited expression
of a gene with a complementary mRNA sequence.
When double-stranded RNAs are introduced into cells,
they are cleaved into short double-stranded
molecules (short interfering RNAs or siRNAs) by an
enzyme called Dicer.
The siRNAs associate with a complex of proteins
known as the RNA-induced silencing complex (RISC),
where mRNA is cleaved.
 Direct inhibition of protein functions
It is sometimes possible to interfere directly
with the function of proteins.

Functional interfering antibody Dominant inhibitory mutants

And so on……….