Techniques used for elucidation

of cell membrane Structure

• Lipid bilayer is the major site of protein lipid
interaction. Information related to structural
organization and function of biological membrane
can be studied by a number of fluorescence
techniques, microscopic methods ,single particle
analysis, centrifugation, live cell imaging methods
etc.
• Because of their hydrophobic transmembrane
domains, integral membrane proteins are
difficult to isolate in a soluble form.
• Removal of these proteins from the
membrane normally requires the use of a
detergent, such as the ionic (charged)
detergent SDS (which denatures proteins) or
the nonionic (uncharged) detergent Triton X‐
100 (which generally does not alter a protein’s
tertiary structure)
Microscopes
• Most broadly, light microscopy techniques can be divided into
two categories: brightfield and fluorescence.
• In brightfield microscopy, the light source and detection
objective are placed on opposite sides of the sample, and the
sample is imaged by its effect on the light passing through it as
the sample absorbs, scatters, or deflects the light.
• Because most cells are thin and transparent, they do not absorb
much light and so are difficult to see without adding optics that
allows the phase shift of light induced by the cells to be seen.
• The two most commonly used techniques to visualize this
phase shift are phase contrast, which causes cells to appear
dark on a light background, and differential interference
contrast (DIC), which gives a pseudo–three-dimensional (3D)
shaded appearance to cells 
 FLUORESCENCE MICROSCOPY
• Fluorescence microscopy uses fluorescent dyes
(fluorophores), which are molecules that absorb one
wavelength of light (the excitation wavelength) and emit a
second, longer wavelength of light (the emission
wavelength).
• Most molecules in the cell are not very fluorescent, so
fluorescent labels to be imaged are typically introduced by
the experimenter.
• This allows the labels to be targeted to the molecule(s) of
interest, either by genetically encoding a fluorescent
protein or by binding a fluorescently labeled antibody.
• The beam of monochromatic light is focused on the
specimen containing the fluorophore, which becomes
excited and emits light of a visible wavelength that is
focused by the objective lens into an image that can be
• seen by the viewer or digitally captured using a charge‐
coupled device (CCD).
• Fluorescence microscopy is typically done using
epifluorescence, in which the fluorescence excitation
light illuminates the sample through the same objective
that is used to detect the emission from the sample.
• A fluorescence filter cube separates the light by
wavelength so that the emitted light can be imaged
without interference from the excitation light.
• The two major techniques for introducing fluorescent
labels into cells are immunofluorescence, in which
fluorescently labeled antibodies that bind to specific
proteins in cells are introduced, and genetic
introduction of a fluorescent protein.
• In immunofluorescence, the cells are first fixed to
cross-link proteins in the cell and then permeabilized
to allow antibodies access to the cellular environment.
• Typically, primary antibodies, which recognize the
proteins of interest in the cell, are first introduced.
After unbound antibodies are washed off, fluorescently
labeled secondary antibodies, which bind to the
primary antibodies, are added. This is known as
indirect immunofluorescence and makes it easy to
switch primary antibodies, as they do not need to be
labeled, and the secondary antibodies typically have
broad specificity (e.g., a goat anti-mouse secondary
that recognizes all mouse immunoglobulin Gs).
• Genetic introduction of a fluorescent protein
involves fusing a fluorescent protein to a target of
interest, which is then either introduced into the
genome of the cell or expressed from a plasmid.
• This allows imaging of proteins in live cells and, if
done by genomic introduction, means that the
protein is expressed from its endogenous
promoter at its endogenous level.
• For both immunofluorescence and fluorescent
protein imaging, it is straightforward to image
four colors in a cell. 
• Membrane probes
• Membrane probes include fluorescent analogs of
natural lipids, as well as lipophilic organic dyes that
have little structural resemblance to natural
biomolecules. using spectroscopic and/or microscopic
experimental approaches.
• These probes are used for structural and biophysical
analysis of membranes, for following lipid transport
and metabolism in live cells.
• The basic structural unit of biological membranes is the
phospholipid bilayer, and because the vast majority of
naturally occurring membrane lipids are non-
fluorescent, extrinsic membrane probes are widely
used.
• Two major issues arise regarding the use of extrinsic
fluorescent probes in membrane studies. First, the
behavior of the probe molecules inside the bilayer (what
transverse region of the bilayer is the probe sensitive to,
its translational and rotational dynamics) is often not fully
understood.
• Second, when interpreting the results of fluorescence
experiments, it can be hard to distinguish between
legitimate membrane properties and perturbing effects
resulting from probe incorporation. 
• Molecular dynamics (MD) simulations can provide detailed
atomic-scale information, and have been extensively used
in the study of lipid bilayer structure and dynamics
• Fluorescence Recovery after Photobleaching (FRAP)
• FRAP is a technique is based on the photobleaching property
of fluorophores.
• The basic apparatus comprises an optical microscope, a light
source and some fluorescent probe. 
• The bilayer is uniformly labeled with a fluorescent tag. A
background image of the sample is saved before
photobleaching.
• Region of interest is selectively photobleached by a high laser
power.
• Then, the ROI is observed for fluorescence recovery, caused
by diffusion, interactions or reactions of the surrounding
fluorophores.
• This diffusion proceeds in an ordered fashion, and is
analytically determinable.
• Finally it yields a recovery curve. According to the
steepness of the recovery, diffusion coefficients,
binding rates or turnover rates can be determined.
• If after some time the fluorescence doesn't reach
the initial level anymore, then some part of the
fluorescence is caused by an immobile fraction
(that cannot be replenished by diffusion).
• If the fluorescent proteins bind to static cell
receptors, the rate of recovery will be retarded.
• Because photobleaching is an irreversible process,
immobile molecules will not recover at all.
• This is most widely exploited to investigate protein
binding.
• Applications
• Used to study the mobility of individual lipid molecules within a cell
membrane, membrane heterogenecity and the diffusion of
proteins in the membrane.
• Used in association with GFP to track the membrane proteins,
protein interaction patterns and the protein binding.
• TIR-FRAP is used to calculate the rates of binding and unbinding of
hormones to and from the cell surface.

Single‐particle tracking (SPT)

• In this individual membrane protein molecules are labeled, usually
with fluorescent molecular tags that emit light under a microscope.
• The movements of the labeled molecules are then followed by a
type of microscopy known as TIRF (Total Internal Reflection
Fluorescence) that is specialized for imaging fluorescent molecules
at the surface of cells.
• Application:
• SPT is used to reveal information on the diffusion of
molecules in a membrane, their interaction with other
particles etc.
• It is a suitable technique to investigate the diffusion
characteristics of lipid or membrane-attached proteins.
• This technique has been applied to investigate the motion
of lipids and proteins within membranes, molecules in the
nucleus / cytoplasm / organelles and molecules in lipid
granules, vesicles, and particles introduced in the
cytoplasm or the nucleus.
• Phase‐Contrast Microscopy
• The phasecontrast microscope makes highly
transparent objects more visible We can distinguish
different parts of an object because they affect light
differently from one another.
• One basis for such differences is refractive index. Cell
organelles are made up of different proportions of
various molecules: DNA, RNA, protein, lipid,
carbohydrate, salts, and water. R
• egions of different composition are likely to have
different refractive indices.
• Normally such differences cannot be detected by our
eyes.
• The phase‐contrast microscope converts differences in
refractive index into differences in intensity (relative
brightness and darkness), which are visible to the eye.
• Phase‐contrast microscopes accomplish this result by (1)
separating the direct light that enters the objective lens
from the diffracted light emanating from the specimen
and (2) causing light rays from these two sources to
interfere with one another.
• The relative brightness or darkness of each part of the
image reflects the way in which the light from that part
of the specimen interferes with the direct light.
• Phase‐contrast microscopes are most useful for
examining intracellular components of living cells at
relatively high resolution.
• For example, the dynamic motility of mitochondria,
mitotic chromosomes, and vacuoles can be followed
and recorded with these optics The phase‐contrast
microscope has optical handicaps that result in loss
of resolution, and the image suffers from interfering
halos and shading produced where sharp changes in
refractive index occur. The phase‐contrast
microscope is a type of interference microscope .
• Electron microscopy
• Electron microscopy can be used to study
membrane protein structure. This provides a
snapshot of the naturally occurring conformation
of individual proteins in the bilayer.
• Transmission electron microscopes
• ( TEMs ) form images using electrons that are
transmitted
• through a specimen, whereas scanning electron
microscopes ( SEMs )
• utilize electrons that have bounced off the
surface of the specimen
• Freeze etching and Freeze fracture
• The concept that proteins penetrate through membranes, rather
than simply remaining external to the bilayer, was derived
primarily from the results of freeze fracture technique.
• Method involves freezing the tissue at -130°C in liquid freon.
Tissue is then transferred in to an evacuated chamber at -100°C.It
contains a knife fixed to a microtome used for cutting or fracturing
or cracking the tissue.
• Once fractured the sample is left in a vaccum for evaporation of
water from exposed surface. This process is called freeze etching.
• At the cut surface a coating of mixed platinum and carbon is made
at an angle so that a layer of it is made on the material according
to its contour.
• Additional coating of carbon is made to provide enough support
to the material cast. The coating along with the tissue is removed
from the chamber and is made to float on a material like acid, that
will remove the tissue
• A replica of carbon-platinum deposition is left
behind. Replica is then used for study under the TEM.

• Electrons can easily pass through the carbon but

platinum is electron dense and therefore appears
black in the electron micrograph.
• The micrograph produced by this technique gives a 3-
D impact on the material.
• X‐ray crystallography (or X‐ray diffraction )
• Utilizes protein crystals, which are bombarded with a fine beam of
X‐rays.
• The radiation that is scattered (diffracted) by the electrons of the
proteins atoms strikes an electron‐sensitive detector placed behind
the crystal.
• The diffraction pattern produced by a crystal is determined by the
structure within the protein; the large number of molecules in the
crystal reinforces the reflections, causing it to behave as if it were
one giant molecule.
• The positions and intensities of the reflections, can be related
mathematically to the electron densities within the protein,
because it is the electrons of the atoms that produced them.
• The resolution obtained by X‐ray diffraction depends on the
number of spots that are analyzed.
• Myoglobin was the first protein whose structure was determined
by X‐ray diffraction.
 • Radioisotopes autoradiography in structure elucidation
• Atoms having the same number of protons and different
numbers of neutrons are said to be isotopes of one another.
• Isotopes are radioactive when they contain an unstable
combination of protons and neutrons. Atoms that are
unstable tend to break apart, or disintegrate, thus achieving
a more stable configuration.
• When an atom disintegrates, it releases particles or
electromagnetic radiation that can be monitored by
appropriate instruments.
• The radioisotopes of greatest importance in cell
biologicalresearch.
• Three main forms of radiation can be released by atoms
during their disintegration: alpha particle, beta particle,
gamma radiation.
• The most commonly used isotopes are beta emitters, which
are monitored by either of two different methodologies:
liquid scintillation spectrometry or autoradiography.
• Autoradiography is a broad‐based technique used to
determine where a particular isotope is located, whether in
a cell, in a polyacrylamide gel, or on a nitrocellulose filter.
• Autoradiography takes advantage of the ability of a particle
emitted from a radioactive atom to activate a photographic
emulsion, much like light or X‐rays activate the emulsion
that coats a piece of film.
• If the photographic emulsion is brought into close contact
with a radioactive source, the particles emitted by the
source leave tiny, black silver grains in the emulsion after
photographic development.
• Autoradiography is used to localize radioisotopes within tissue
sections that have been immobilized on a slide or TEM grid.
• The steps involved in the preparation of a light microscopic
autoradiograph are as follows.
• The emulsion is applied to the sections on the slide or grid as a
very thin overlying layer, and the specimen is put into a
lightproof container to allow the emulsion to be exposed by
the emissions.
• The longer the specimen is left before development, the
greater the number of silver grains that are formed.
• When the developed slide or grid is examined in the
microscope, the location of the silver grains in the layer of
emulsion just above the tissue indicates the location of the
radioactivity in the underlying cells.