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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