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REVEALING THE CELL’S SECRETS
O
ur DNA might provide the blueprint for how
to build our bodies, but it is the proteins
that really do the heavy lifting. While there
are around 20,000 genes encoded in
our DNA, the total number of proteins is
estimated to be many times more—possibly
as many as a million*. This is because a single gene might
produce multiple variants of a particular protein through,
for example, alternative splicing of the messenger RNA.
Posttranslational modification of the nascent protein, such
as phosphorylation and glycosylation, may also significantly
or subtly change its function, yielding many possible
functional protein variants.
Understanding how a particular DNA sequence gives
rise to a particular protein provides us with some insight
into that protein, but a deeper investigation of how the
protein is made, where it is located, and how much of it is
present in different cell types is required to enable a true
understanding of its function. As mentioned above, changes
to the protein soon after its translation can impact its final
function, as well as its final location. Conversely, its location
may also affect its function. Intensive study has therefore
gone into finding out as much as possible about the lifecycle
of proteins.
EXPL
the pathways and processes in which these proteins are
involved. Fourteen compartments are covered, including
mitochondria, the cytosol, and the nucleoplasm. The
description for each compartment hyperlinks the reader
back to the free Protein Atlas resource, where a rich trove
of data resides, open for exploration by anyone—from
serious researchers to those who may just have an interest
in cell biology.
Alongside this information, the poster includes a helpful
timeline that highlights important milestones in the history
of microscopy, from the first compound microscope
in the late 1500s to more recent developments in
superresolution imaging. Paralleling that timeline is another
outlining the important discoveries in cell biology. All of
these advances made it possible for us to be where we
are today—finally beginning to unravel the complexity of
the proteome and gaining a clearer understanding of how
our DNA renders our proteins, and how those proteins are
organized, interact, and ultimately define who and what we
are as human beings.
Interested in the human cell?
Open this poster to learn about the most
detailed mapping of the human cell ever
done. The Human Protein Atlas project is
presenting a high-resolution map of the
human cell. The proteins have been
localized with high precision to cellular
organelles, structures and sub-structures,
with high-resolution images freely available
for you to explore.
Sean Sanders, Ph.D.
Editor, Custom Publishing,
Science
The study of the entire complement of human proteins
is known as proteomics. Researchers in this field seek
to identify and characterize each and every protein in a
specific location—be it the whole body or a specific tissue. A
previous Science poster† looked predominantly at the tissue
proteome, highlighting how tissues have unique patterns of
protein expression and how this pattern might be disrupted
by dysfunction and disease.
The antibodies used are Triple A Polyclonals
provided by Atlas Antibodies.
MADE IN SWEDEN
The poster that you are now viewing aims to delve down
an additional layer into the cellular proteome, taking a
journey into the subcellular compartments within a single
cell to discover what secrets each organellar proteome
might hold. Characterizing the distribution of individual
proteins at a subcellular level provides important clues to
RE
*proteomics.cancer.gov/whatisproteomics
†poster.sciencemag.org/humanproteome
Roger Goncalves, Sales Manager
Custom Publishing
Europe, Middle East, and India
[email protected]
Writers: Mikaela Wiking, M.Sc.; Tove Alm, Ph.D.; Mathias Uhlen, Ph.D.; Emma Lundberg, Ph.D.
Illustrator/Designer: Luca Marziani
Editor: Sean Sanders, Ph.D.
+41-43-243-1358
Editor: Sean Sanders, Ph.D.
Writers: Mikaela Wiking, M.Sc.; Tove Alm, Ph.D.;
Mathias Uhlen, Ph.D.; Emma Lundberg, Ph.D.
Illustrator/Designer: Luca Marziani
Sponsored by
Publication date: 31 October, 2014
Produced by the Science/AAAS
Custom Publishing Office
www.antibodypedia.com
Immunocapture is a method
that uses an antibody to isolate a
protein from a solution. When coupling
this technique with mass spectrometry (MS),
the proteins captured by the antibody can be
identified. The peptides for the target protein
should be on the top of the generated
peptide list in order for the antibody
to be considered specific.
Tagging proteins on the
genetic level with an affinity tag or
a fluorescent protein can be used to
validate the antibody for the target protein.
Tagged proteins should preferably be expressed
at endogenous levels. The expression
pattern of the tag should overlap
with the expression pattern created
when using the antibody for
protein detection.
IMMUNOCAPTURE MS
TAGGED
PROTEINS
Percentage utilization of antibodies in listed applications according to data in Antibodypedia. Western blotting (WB) is the
most commonly used application, followed by immunohistochemistry (IHC), immunocytochemistry (ICC), and flow sorting (FS).
Other
5%
INDEPENDENT
ANTIBODIES
ORTHOGONAL
STRATEGIES
A
M
S
E
K
IHC
29%
WB
51%
Reverse phase protein
arrays (RPPA)
Immunoprecipitation (IP)
Sandwich assays (SA)
Flow sorting (FS)
Immunocytochemistry
(ICC)
Immunohistochemistry
(IHC)
Western blotting (WB)
Application
Genetic
Strategies
Orthogonal
Strategies
Independent
Antibodies
Tagged
Proteins
Immunocapture
Mass Spectrometry
PILLAR
FIVE CONCEPTUAL PILLARS FOR ANTIBODY VALIDATION. Antibodies are powerful tools used in many different
applications to detect proteins. The power of the antibody lies in its ability to recognize a specific target. It is crucial to
properly validate the antibody for binding to its intended target, to test the antibody in the intended application, and to
understand the context where it will be used. The five pillars for antibody validation are summarized below.
HOW TO TARGET VALIDATE
YOUR ANTIBODY
TI W
E
R
U
THE GUIDELINES
K
R
O
Due to the need for properly
validated antibodies, the International
Working Group on Antibody Validation
(IWGAV) has made an effort to standardize
best practices, resulting in a publication
proposing “Conceptual Pillars for
Validation of Antibodies.” The pillars
presented here are directed to
both users and producers
of antibodies.
N
I
S
U
O
Y
A
R
L
P
P
GENETIC
STRATEGIES
ANTIBODYPEDIA
T
A
IC
!
N
IO
Independent antibody strategies
use two or more antibodies
recognizing different epitopes
(binding sites) on the target protein. This
method minimizes the likelihood of off-target
binding to the same unrelated protein.
Antibody validation is achieved when the
unique antibodies give comparable
results when using the same
detection method.
Genetic strategies can be used
to generate genetically modified
samples where the target protein is
knocked out or knocked down. This method
provides a direct link between the gene and
the target protein. The antibody is considered
validated for its target when the signal from
the original sample is significantly
downregulated in the genetically
modified sample.
ICC
9%
This table summarizes for which applications the five conceptual pillars are recommended. The represents support for
the pillar in the application. Ref.: M. Uhlen et al., A proposal for validation of antibodies. Nat. Methods 13, 823–827 (2016).
Orthogonal strategies compare
an antibody-based method with
an antibody-independent method, for
example targeted proteomics approaches
using labeled internal standards. Identifying
and measuring your target protein in a set
of samples with a method not involving
antibodies should give comparable
results to the antibody-based
method.
The Antibodypedia database
lists antibodies provided by
academia and commercial companies.
Antibodypedia ranks antibodies based
on the amount and quality of the knowledge
associated with them, putting the antibody with
the most information available on top of the
search list, and assisting you in selecting
the most appropriate antibody
for your experiment.
FS
6%
ma
ke
su
The guidelines
re
wo
rk
si
Genetic
strategies
Five conceptual pillars for antibody validation. Antibodies are powerful tools used in many different
applications to detect proteins. The power of the antibody lies in its ability to recognize a specific target. It is crucial to
properly validate the antibody for binding to its intended target, to test the antibody in the intended application, and to
understand the context where it will be used. The five pillars for antibody validation are summarized below.
ny
Antibodypedia
it
Due to the need for properly
validated antibodies, the International
Working Group on Antibody Validation
(IWGAV) has made an effort to standardize
best practices, resulting in a publication
proposing “Conceptual Pillars for
Validation of Antibodies.” The pillars
presented here are directed to
both users and producers
of antibodies.
How to target validate
your antibody
ou
Genetic strategies can be used
The Antibodypedia database
to generate genetically modified
lists antibodies provided by
samples where the target protein is
academia and commercial companies.
knocked out or knocked down. This method
Antibodypedia ranks antibodies based
provides a direct link between the gene and
on the amount and quality of the knowledge
the target protein. The antibody is considered
associated with them, putting the antibody with
validated for its target when the signal from
the most information available on top of the
the original sample is significantly
search list, and assisting you in selecting
downregulated in the genetically
the most appropriate antibody
Orthogonal
Independent
modified sample.
for your experiment.
ra
Pillar
pp
Immunocapture is a method
that uses an antibody to isolate a
protein from a solution. When coupling
this technique with mass spectrometry (MS),
the proteins captured by the antibody can be
identified. The peptides for the target protein
should be on the top of the generated
peptide list in order for the antibody
to be considered specific.
n!
Tagging proteins on the
genetic level with an affinity tag or
a fluorescent protein can be used to
validate the antibody for the target protein.
Tagged proteins should preferably be expressed
at endogenous levels. The expression
pattern of the tag should overlap
with the expression pattern created
when using the antibody for
protein detection.
Immunocapture MS
io
at
antibodies
Independent antibody strategies
use two or more antibodies
recognizing different epitopes
(binding sites) on the target protein. This
method minimizes the likelihood of off-target
binding to the same unrelated protein.
Antibody validation is achieved when the
unique antibodies give comparable
results when using the same
detection method.
Orthogonal
Strategies
Independent
Antibodies
Tagged
Proteins
Immunocapture
Mass Spectrometry
Western blotting (WB)
li c
Tagged
proteins
strategies
Orthogonal strategies compare
an antibody-based method with
an antibody-independent method, for
example targeted proteomics approaches
using labeled internal standards. Identifying
and measuring your target protein in a set
of samples with a method not involving
antibodies should give comparable
results to the antibody-based
method.
Genetic
Strategies
Application
Immunohistochemistry
(IHC)
Immunocytochemistry
(ICC)
Flow sorting (FS)
Sandwich assays (SA)
Immunoprecipitation (IP)
Reverse phase protein
arrays (RPPA)
This table summarizes for which applications the five conceptual pillars are recommended. The represents support for
the pillar in the application. Ref.: M. Uhlen et al., A proposal for validation of antibodies. Nat. Methods 13, 823–827 (2016).
Other
5%
FS
6%
ICC
9%
IHC
29%
WB
51%
Percentage utilization of antibodies in listed applications according to data in Antibodypedia. Western blotting (WB) is the
most commonly used application, followed by immunohistochemistry (IHC), immunocytochemistry (ICC), and flow sorting (FS).
www.antibodypedia.com