The Human Protein Atlas Portal
The Human Protein Atlas is a genome-wide project with a primary goal of producing a complete localization map of the human proteins. Today the Human Protein Atlas contains protein localization data for 12 238 human genes corresponding to more than 60% of the human genome. The proteins have been mapped by using over 15 598 different antibodies. Each antibody has been used for immunohistochemical staining of 48 normal human tissue samples in triplicates and 432 human cancer samples covering the 20 most common cancer types. In addition, 59 cells and cell lines has been immunohistochemically stained and annotated for each antibody. In total, close to 700 immunohistochemistry images are available for each antibody.
The Human Protein Atlas also contains subcellular localization data, achieved by confocal microscopy analysis, for approximately 8800 proteins and supportive Western Blot results for 6800 antibodies.
Samples from cancer are also derived from surgical material. The inclusion of tumors has been based on availability and representativity. Due to subgroups and heterogeneity of tumors within each cancer type, included cases represent a typical mix of specimens from surgical pathology. However, an effort has been made to include high and low grade malignancies where such is applicable. Tumor heterogenity and inter-individual differences is also reflected in diverse expression of proteins resulting in variable immunohistochemical staining patterns.
As a complement to the representation of normal and cancer tissue, the Human Protein Atlas displays images of a selection of widely used and well characterized human cell lines as well as cell samples from normal individuals and leukemia/lymphoma patients. A cell microarray has been used to enable immunohistochemical staining of a panel of cell lines and cell samples. Duplicates from 47 cell lines and 12 samples of primary blood cells renders a total of 118 cell images per antibody. All cells are fixed in 4% paraformaldehyde prior to paraffin embedding and immunohistochemical staining.
In addition to antibody staining, the cells are also stained with cellular probes targeting specific compartments in the cells in order to facilitate the annotation of the subcellular localization of the protein targeted by the Atlas Antibody. The following probes/organelles are used as references; (i) DNA stain DAPI for the nucleus, (ii) tubulin as internal control for fixation quality and homogeneity, and (iii) calreticulin for the endoplasmatic reticulum (ER).
The resulting confocal images are single slice images representing one optical section of the cells. The microscope settings are adjusted for each sample to always use the full dynamic range of the detector. The different organelle probes are displayed as different colors/channels in the multicolor images; the antibody staining is shown in green, nuclear stain in blue, micro-tubules in red and ER in yellow.
Antibodies included in the Human Protein Atlas are analyzed using a standardized protocol in a single attempt without further efforts to optimize the procedure and therefore it cannot be excluded that certain observed binding properties are due to technical rather than biological reasons and that further optimization could result in a different outcome.
A selection of antibodies is currently being tested in over-expressed lysates (Origene Technologies).
The Human Protein Atlas also contains subcellular localization data, achieved by confocal microscopy analysis, for approximately 8800 proteins and supportive Western Blot results for 6800 antibodies.
Immunohistochemistry
Tissue microarrays provides the possibility to immunohistochemically stain a large number and variety of normal and cancer tissues. Samples have been collected from anonymized paraffin embedded material of surgical specimens, in accordance with approval from the local ethics committee. The specific binding of an antibody to its corresponding antigen results in a brown-black staining. The tissue section is counterstained with hematoxylin to enable visualization of microscopical features. Hematoxylin staining is unspecific and results in a blue coloring of both cells and extracellular material.Samples from cancer are also derived from surgical material. The inclusion of tumors has been based on availability and representativity. Due to subgroups and heterogeneity of tumors within each cancer type, included cases represent a typical mix of specimens from surgical pathology. However, an effort has been made to include high and low grade malignancies where such is applicable. Tumor heterogenity and inter-individual differences is also reflected in diverse expression of proteins resulting in variable immunohistochemical staining patterns.
As a complement to the representation of normal and cancer tissue, the Human Protein Atlas displays images of a selection of widely used and well characterized human cell lines as well as cell samples from normal individuals and leukemia/lymphoma patients. A cell microarray has been used to enable immunohistochemical staining of a panel of cell lines and cell samples. Duplicates from 47 cell lines and 12 samples of primary blood cells renders a total of 118 cell images per antibody. All cells are fixed in 4% paraformaldehyde prior to paraffin embedding and immunohistochemical staining.
Immunofluorescence
As a complement to the immunohistochemically stained cells and tissues, the Human Protein Atlas displays high resolution, multicolor images of immunofluorescently stained cells. This provides spatial information on protein expression patterns on a fine cellular and subcellular level. Three cell lines, U-2OS, A-431 and U-251MG, originated from different human tissues have been chosen to be included in the immunofluorescent analysis. To enhance the probability for a large number of proteins to be expressed, cell lines were selected from different lineages, i.e. tumor cell lines from mesenchymal, epithelial and glial tumors. The selection was furthermore based on morphological characteristics, widespread use and multitude of publications using these cell lines.In addition to antibody staining, the cells are also stained with cellular probes targeting specific compartments in the cells in order to facilitate the annotation of the subcellular localization of the protein targeted by the Atlas Antibody. The following probes/organelles are used as references; (i) DNA stain DAPI for the nucleus, (ii) tubulin as internal control for fixation quality and homogeneity, and (iii) calreticulin for the endoplasmatic reticulum (ER).
The resulting confocal images are single slice images representing one optical section of the cells. The microscope settings are adjusted for each sample to always use the full dynamic range of the detector. The different organelle probes are displayed as different colors/channels in the multicolor images; the antibody staining is shown in green, nuclear stain in blue, micro-tubules in red and ER in yellow.
Western Blot
Western Blot analysis of antibody specificity is performed using a routine sample setup composed of IgG/HSA-depleted human plasma and protein lysates from a limited number of human tissues and cell lines. Antibody binding is visualized by chemiluminescence detection in a CCD-camera system using a peroxidase (HRP) labelled secondary antibody.Antibodies included in the Human Protein Atlas are analyzed using a standardized protocol in a single attempt without further efforts to optimize the procedure and therefore it cannot be excluded that certain observed binding properties are due to technical rather than biological reasons and that further optimization could result in a different outcome.
A selection of antibodies is currently being tested in over-expressed lysates (Origene Technologies).