The Executive Management is responsible for the overall strategies of the consortium and for managing the overall program:
Prof Mathias Uhlen, Program Director
Assoc prof Emma Lundberg, Director of Cell Atlas
Dr Cecilia Lindskog Bergström, Director of Tissue Atlas
Prof Fredrik Ponten, Director of Pathology Atlas
Prof Peter Nilsson, Head of protein arrays technologies
Prof Sophia Hober, Head of protein science
Kalle von Feilitzen, Director of Bioinformatics and IT
Inger Åhlen, Administrative Coordinator
AlbaNova site, Stockholm
Antigen and Antibody Factory
Group leader: Dr Hanna Tegel
Personnel: Delaram Afshari (research engineer), Anna Berling (team leader), Melanie Dannemeyer (researcher), Siri Ekblad (research engineer), Henric Enstedt (research engineer), Sara Kanje (researcher), Roxana Mihai (laboratory engineer), Muna Muse (laboratory engineer), Anne-Sophie Svensson (team leader), Malin Westin (laboratory engineer), Lan Lan Xu (laboratory engineer)
Responsibility: (i) Production of recombinant PrEST expression clones including cDNA synthesis, cloning, and
plasmid purification. (ii) Production and purification of PrEST-proteins used for preparation of
antigens and affinity columns. (iii) Management of immunization outsourcing. (iv) Generation of
purified antibodies through affinity purification of polyclonal antisera. (v) Western blot (WB) analysis
of antibodies approved in protein array analysis. (vi) WB antibody validation using over-expression
lysates. (vii) Storage and distribution of antigens and antibodies within the program.
Description: PrEST regions are first amplified with RT-PCR from a total RNA template pool with the specific
oligonucleotide primers designed in the Bioinformatics module. Three different RNA pools are used,
two consisting of total RNA from six individual human tissues, and one comprising total RNA from
10 different cell lines. For PrESTs not found in any RNA pool, cDNA clones are used as template
(Ultimate™ ORF LITE Clones (Human collection), Life Technologies, Carlsbad, CA and Mammalian
Gene Collection (The MGC Project team, Genome Res. 2009)). Amplicons are automatically
processed with solid phase restriction, and ligated into the plasmid vector pAff8c (Larsson, M. et al,
2000) where the human gene fragment is fused to a histidine tag and albumin binding protein
(His6ABP). After transformation into E. coli Rosetta(DE3), (His6ABP). After transformation into E. coli Rosetta(DE3), inserts are verified by DNA sequencing to
omit clones with mutations and approved clones are single cell streaked. Plasmids are collected from
all purified clones for deposition in the clone library and glycerol stocks are prepared and used as
starting material for protein production.
All proteins are expressed as His6ABP fusions in E. coli shake flask cultures upon
induction with IPTG. A fully automated protein purification system has been
developed to allow for purifications of up to 60 cell lysates at a time. One-step purification is enabled
by the hexahistidine affinity tag and metal affinity chromatography (IMAC) and performed under
denaturing conditions. After evaluation of protein concentration and purity, the molecular weight of
the PrEST proteins is determined by mass spectrometry as a final quality control. The purified proteins
are then used to prepare antigens and affinity columns with PrEST-ligands. In addition, affinity resin
with His6ABP-ligand is
The group is also responsible for the generation, purification and Western Blot analysis of all
antibodies produced in the Human Protein Atlas (HPA) program. Polyclonal antisera generated
together with collaborative partners are carefully purified in a three-step fashion consisting of:
depletion of unwanted specificity, capture of wanted specificity and a final buffer exchange step. A
manual process using gravity-flow columns carries out depletion of antibodies with unwanted
specificity. The following steps are performed on the ÄKTAxpress chromatography system enabling a
high-throughput semi-automated process where captured antibodies are eluted by a low pH glycine
buffer and automatically loaded onto a desalting column for buffer exchange. Antibodies are
supplemented with 50% glycerol and 0.02% sodium azide for long-term storage at -20°C. The binding
specificity of all antibodies is determined on protein microarrays to certify that only antibodies with
high specificity and low background binding are approved for immunohistochemistry analysis. All
approved antibodies are further analyzed in a high-throughput WB platform using protein lysates from
human cell lines (RT-4 and U-251 MG), human plasma depleted of IgG and HSA and whole tissue
lysates from human liver and tonsil. A selection of the published antibodies, initially scored as uncertain in the standard
WB panel, have been revalidated in a WB set-up comprising an over-expression lysate
(VERIFY Tagged Antigen™, OriGene Technologies, Rockville, MD) as a positive control.
ABP - Albumin Binding Protein
IPTG - Isopropyl-B-D-Thiogalactopyranoside
IMAC - Immobilized Metal Affinity Chromatography
Epitope Mapping and Therapeutic Antibodies
Group leader: Asstn. Prof. Johan Rockberg
Personnel: Francis Jingxin Hu (PhD-student), Magnus Lundqvist (PhD-student), Anna-Luisa Volk
(PhD-student), Niklas Thalen (PhD-student), Mona Moradi (PhD-student)
Responsibility: (i) To determine antibody epitopes for HPA antibodies, and(ii) to generate
monoclonal reagents towards selected targets and screen them in functional assays for therapeutic effect.
Description: Epitope mapping is performed on a selection of the HPA's monospecific
and monoclonal reagents using a combination of platforms including: (i) cell surface display
of target directed peptide libraries using staphylococcal display, (ii) suspension bead arrays
for mapping using overlapped or alanine mutated synthetic peptides, and (iii) large scale synthetic peptide arrays.
Monoclonal antibodies to clinically relevant target proteins are selected using a combination
of phage display and cell surface display of antibody fragments. These are subsequently screened
for desired effect in cell-based assays and cloned for production in mammalian cells as full-length
Group leader: Prof Sophia Hober
Personnel: Sara Kanje (researcher), Sarah Lindbo (PhD-student),
Emma von Witting (PhD-student), Julia Scheffel (MSc-student)
Responsibility: Protein science research.
Description: The module coordinates and conducts research projects aiming to extend the scientific
outcome of data generated within the Human Protein Atlas project as well as improving
current methodology. Both biological and technical research projects based on the vast
amount of data generated are performed to further explore the function, localization and
interactions of human proteins.
SciLifeLab site, Stockholm
Protein Array Technologies
Group leader: Prof Peter Nilsson
Personnel: Ronald Sjöberg (researcher), Anna Månberg (researcher), Elisa Pin (researcher), Eni Andersson (research engineer), Cecilia Hellström (research engineer), Jennie Olofsson (research engineer), August Jernbom Falk (research engineer), David Just (PhD Student), Julia Remnestål (PhD Student), Sofia Bergström (PhD Student), Maria Mikus (postdoc), Philippa Reuterswärd (postdoc)
Responsibility: To validate the specificity and selectivity of all purified HPA antibodies. To develop and utilize peptide, antigen and antibody based microarray methodologies for large scale analysis of body fluids in the context of biomarker discovery and autoantibody profiling.
Description: Methodology for microarray based analysis of antibody specificity has been developed, where all purified
antibodies are analyzed on protein arrays with immobilized PrESTs. Each microarray is divided into 21 replicated subarrays with
384 PrESTs, enabling the analysis of 21 antibodies simultaneously. The antibodies are detected through a fluorescently labeled
secondary antibody. A specificity plot is generated for each antibody, where the signal from the binding to its antigen is
compared to the unspecific binding to all the other PrESTs. A dual color system is used in order to verify the presence of
the spotted PrESTs. Several complementary microarray formats for systematic analysis of body fluids are being utilized and under
constant development. The PrEST-arrays have been implemented for systematic antigen-based plasma profiling for the screening of
new autoimmunity components. The antibody microarrays with the possibility for simultaneous analysis of large amounts of
analytes with high sensitivity and the reverse phase serum microarrays which enable serum from very large patient cohorts to
be analyzed simultaneously are both utilizing in-house produced planar microarrays. The main platform for systematic
antibody-based plasma profiling, is although the suspension bead array format with capacity for multiplexing in two dimensions,
enabling the simultaneous profiling of 384 antibodies on 384 samples, see Plasma profiling.
Director: Assoc prof Emma Lundberg
Group leader Cell Atlas: Dr. Peter Thul
Personnel: Martin Hjelmare (technology and development manager), Dr. Charlotte Stadler (head of Cell Profiling facility), Dr. Ulrika Axelsson (research engineer), Anna Bäckström (research engineer), Dr. Frida Danielsson (Post-doc), Jenny Fall (technician), Christian Gnann (research engineer), Diana Mahdessian (PhD-student), Dr. Anna Martinez Casals (research engineer), Rutger Schutten (research engineer), Dr. Devin Sullivan (post-doc), Casper Winsnes (PhD-student), Dr. Hao Xu (researcher), Lovisa Åkesson (PhD-student)
Responsibility: Creation of the HPA Cell Atlas: (i) Determine the subcellular distribution of proteins using high-resolution confocal microscopy, (ii) validation of antibody specificity using gene editing and silencing technologies (iii) annotation and knowledge- based curation of subcellular distribution profiles in the Cell Atlas.
Description: The Cell Profiling group is responsible for determination of the spatiotemporal subcellular distribution of human proteins. The distribution is systematically assessed at single-cell level using the antibodies generated within the Human Protein Atlas program. Each protein is studied in up to three cell lines, which are selected based on RNA expression levels from a panel of cell lines of different origin. Subcellular distribution is determined by indirect immunofluorescence followed by confocal microscopy. The resulting high-resolution images show the protein of interest as well as markers for the nucleus, microtubule cytoskeleton, and endoplasmic reticulum. This enables manual annotation of protein localization to one or more organelles and subcellular structures, and detection of cell-to-cell variability. The high-resolution confocal images, annotations, and gene expression data can be explored in an interactive gene-centric manner on the Cell Atlas.
The Cell Profiling group uses different strategies for enhanced antibody validation. Most common is validation by independent antibodies, but we also perform genetic validation by knocking down the gene of interest using siRNA, and validation by comparative immunostaining in cells co-expressing a GFP-tagged recombinant version of the protein of interest.
Group leader: Assoc prof Jochen M. Schwenk
Personnel: Eni Andersson (research engineer), Annika Bendes, (research engineer), Sanna Byström (post-doc), Matilda Dale (research engineer), Tea Dodig-Crnkovic (PhD-student), Kimi Drobin (PhD-student), Dr Mun-Gwan Hong (biostatistician), Cecilia Mattsson (research engineer), Ragna Häussler (post-doc), Dr MariaJesus Iglesias (researcher), Gabriella Tekin (research engineer)
Responsibility: Utilizing HPA antibodies in multiplexed affinity assays to profile proteins in human serum and plasma for the discovery, identification and verification potential biomarker candidates.
Description: Antibodies are immobilized on color-coded beads to create antibody arrays in suspension. Each bead array is composed of 384 antibodies that are selected either from defined lists of proteins (targeted design) or by antibody availability (random design). The samples are biotinylated and heat treated for the antibody array analysis, where protein profiles are generated for up to 384 samples at a time.
We are also expanding the profiling procedure into other body fluids (e.g. CSF, BAL or dried blood spots) and are conducting studies such as within different disease areas such as cancer, diabetes, cardiovascular, or neurodegenerative disorders. The data from such multiplexed single-binder assays is accordingly processed and analyzed with biostatistical models to identify which antibodies provide disease indications. Such antibodies then enter further validation assays using mass spectrometry and to develop dual-binder sandwich immunoassays. Subsequent studies are then designed to validate the candidate proteins in larger sample sets from preferably independent collection sites.
Group leader: Dr Jan Mulder
Personnel: Agnieszka Limiszewska (technician), Dr. Nicholas Mitsios (researcher), Dr. Jaekyung Shin (researcher), Evelina Sjöstedt (PhD student), Sania Kheder (technician).
Responsibility: (i) Validation of antibodies against human targets on rodent tissues,
(ii) profiling the distribution of proteins in the developing, adult, and diseased nervous system,
(iii) quantification, annotation, and presentation of whole brain protein distribution profiles.
Description: Mice are widely used in biomedical research and due to its size the mouse brain is very suitable to study regional and cellular protein distributions in the
mammalian nervous system. HPA antibodies against proteins expressed in the mouse nervous system are validated on mouse brain tissue using western blot and immunohistochemistry.
Antibodies that pass validation are used to generate detailed protein distribution profiles using 20-30 coronal sections of the mouse brain with a 400 μm section interval covering all
major brain nuclei. Whole slide immunofluorescence captured at 10x primary objective is analyzed and regional,
cellular and subcellular protein distributions are quantified. Data and images are optimized for online publication.
Group leader: Kalle von Feilitzen
Personnel: Dr Linn Fagerberg (researcher), Mattias Forsberg (research engineer), Fredric Johansson (research engineer), Per Oksvold (research engineer), Lukas Persson (research engineer), Dr Åsa Sivertsson (researcher), Martin Zwahlen (research engineer)
Responsibility: (i) To deliver custom made software solutions for all operations in the Human Protein Atlas project,
(ii) to provide the collected data to the public via the Human Protein Atlas,
(iii) to map and quantify RNA-seq data, and
(iv) to initiate the analysis of human proteins by in silico selection and design of Protein Epitope Signature Tags (PrESTs).
Description: With the LIMS (Laboratory Information Management System) as the backbone, data is collected from each module in the pipeline.
The protein expression profiles, RNA-seq data and raw data from the project is published on the Human Protein Atlas public web site through annual releases.
The Bioinformatics group performs computer-based analysis of protein sequences for selection of protein fragments (PrESTs) to be used as immunogens for generation
of target-specific polyclonal antibodies. A custom made pipeline analyzes RNA-seq data from a large number of tissue, cancer and cell line samples.
The group is also heavily involved in all research performed in the project, such as data collection, data interpretation and visualization as well as statistical analysis.
Site Director: Dr Cecilia Lindskog Bergström
Clinical Director: Prof Fredrik Ponten
Tissue Microarray Production, Immunohistochemistry, and Scanning
Group leader: Ing-Marie Olsson (research engineer)
Personnel: Maria Aronsson (research engineer), Anna-Maria Dénes (research engineer), Jonas Gustafsson (research engineer), Dennis Kesti (biomedical analyst), Lillemor Källström (biomedical analyst)
Responsibility: (i) handling and processing of tissues (biobank material), (ii) handling and storage of antisera, (iii) testing of antibodies, (iv) immunohistochemical staining of tissues slides to be scanned, (iv) scanning of tissue slides and processing images, (v) quality assurance for all areas of responsibility.
Description: Formalin fixed, paraffin embedded tissue specimens are collected from the Department of Pathology, Uppsala University Hospital and subsequently processed for production of tissue microarrays (TMAs), and protein extraction for Western blot analysis. In addition to standard TMAs, additional tissue samples are used for extended profiling of certain proteins. Immunohistochemistry is performed using standardized procedures and includes testing antibodies on TMAs and whole sections of tissues in order to create protein expression profiles. Approved immunostained slides are scanned to generate high-resolution digital images, using a 20x objective. Images are imported to an in-house built software to facilitate subsequent manual scoring of images. All procedures follow strict guidelines and include quality control.
Antibody approval, Protein Profiling, and Antibody Destiny
Group leader: Dr Cecilia Lindskog Bergström
Personnel: Maria Aronsson (research engineer), Anna-Maria Dénes (research engineer), Jonas Gustafsson (research engineer), Borbala Katona (research engineer), Emil Lindström (research engineer), Feria Hikmet Noraddin (research engineer), Charlotte Soläng (research engineer), Lina Thelander (research engineer), Jimmy Vuu (research engineer)
Responsibility: (i) Validation of antibody target specificity (ii),(ii) evaluation of antibodies submitted by commercial vendors and academic scientists, (iii) annotation and final approval of immunohistochemically stained tissues, (iv) determination of antibody reliability based on enhanced antibody validation strategies, (v) generation of knowledge-based protein expression profiles, and (vii) quality assurance of all areas of responsibility.
Description: Optimal antibody dilution and target specificity is assessed by microscopical examination and comparison of immunohistochemical staining with internal and external gene/protein characterization data. For each approved antibody, a final immunostaining protocol is defined and subsequently applied to the standard 8 full-scale TMAs. For extended profiling of certain proteins, also slides containing extended samples are used. Manual annotation of immunohistochemistry is performed using an in-house software, and subsequently curated by an independent second observer. The generated protein profiles then undergo further evaluation and antibodies that pass quality criteria are assigned a reliability score, a knowledge-based protein expression profile and are scheduled for publication in the next version of the Human Protein Atlas.
Group leader: Prof Fredrik Ponten
Personnel: Anna-Maria Dénes (research engineer)
Responsibility: (i) Develop strategies to identify potential biomarkers based on the HPA database and other efforts, (ii) validate proteins that can be used as clinical biomarkers for disease, (iii) participate in clinical studies, collect tumor material and clinical data to generate specific cancer TMAs coupled to clinical databases, (iv) perform statistical analysis and validate the clinical usefulness of identified biomarkers.
Description: The HPA database is actively mined for potential biomarkers with the aim to identify protein expression patterns that could be of medical or biological significance. Projects include various forms of human disease with an emphasis on cancer. Most projects are focused on identification and validation of biomarker candidates that can fulfill currently unmet clinical needs related to diagnostics, prognostics and treatment prediction. To address such questions, patient cohorts representing different cancers are defined and tumor material as well as clinical data is collected. These specifically designed cancer TMAs are produced and used for extended analysis of protein expression patterns to test and validate candidate proteins as useful biomarkers. The biomarker discovery and validation efforts include both internal projects and external collaborative projects.