THE HUMAN PROTEIN ATLAS BLOG

A subcellular map of the human proteome

2017-05-12
Cell Atlas Immunocytochemistry Immunofluorescence Organelle Science

30 organelles and substructures are annotated in the Cell Atlas.

The Cell Atlas database was just recently released with data on more than 12.000 proteins and mapping to 30 organelles. Today, the scientific findings are published in Science in "A subcellular map of the humanproteome".

The cell is a complex entity that carries out multiple functions. In order to do this, the different parts of the cells are organized in structures, called organelles. By investigating the organelles and its proteome, and to understand how the proteins vary over time and space we can truly begin to understand human biology on a detailed level...Read more


Cell Image of the Month – MCM6

2017-02-24
Cell Atlas Image of the week Immunofluorescence Lactase

In 2017, February 28th will be the day that in Sweden is known as "Fettisdagen", (lit. "Fat Tuesday"), in other countries known as "Mardi Gras", "Faschingsdienstag" or "Shrove Tuesday". This day was originally celebrated in the Christian tradition as the last day of a three day feast to prepare for the forty day long fasting period before Easter. Today it has been popularized and in many places around the world this day is now known for its carnivals.

In Sweden together with some other northern European countries we like to eat "semlor" on Fat Tuesday...Read more


Validation of antibodies for bioimaging

2017-01-31
Antibody Antibody Validation Bioimaging Immunofluorescence

Marie Skogs, first author of the study

In a recent publication in Journal of Proteome Research, researchers from the Cell Profiling group of the Human Protein Atlas team present a new approach for validation of antibodies for bioimaging applications.

Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here the researchers present a strategy to systematically validate antibodies for immunofluorescence applications using gene tagging.

Marie Skogs is the first author of the study.

– After a M. Sc...Read more


Sample preparation – planning is crucial

2016-11-22
Antibody Cell Atlas Immunofluorescence

Christian Gnann by the Tecan's Freedom EVO pipetting robot

To prepare all the images for the Cell Atlas, released on December 4, the cells used are primed for staining and microscopy. Sample preparation is an important step when performing immunofluorescence studies. If wrongly applied it can not only cause unsuccessful detection but also generate misleading information.

The sample preparation performed by the Cell Atlas team includes cell fixation, permeabilization, and immunostaining with primary and secondary antibodies.

– The fixation is the crucial step, and different fixation protocols work better for different sets of proteins, Christian Gnann, a research engineer in the Cell Profiling group explains...Read more


Culturing cells for the Human Cell Atlas

2016-11-15
Cell Atlas Immunofluorescence

Anna Bäckström with the cells in a LAF bench

The Human Cell Atlas, to be released on December 4, displays high resolution, multicolour images of immunofluorescently stained cells. This provides spatial information on protein expression patterns on a fine cellular and subcellular level. From the start three cell lines, U-2 OS, A-431 and U-251 MG, originating from different human tissues were chosen to be included in the immunofluorescent analysis...Read more


Image of the week - CDC42 and actin filaments

2016-05-06
Actin Cell cycle Image of the week Immunofluorescence Subcell Atlas

Fig 1. Staining of CDC42EP4 (green) with DAPI (blue) and microtubules (red) in U2 OS cells.

This week HPA image of the week, I've decided to highlight two of my favorite things, the cell cycle and actin filaments!

The protein labeled in Fig 1. is an image of Cell division cycle 42 effector protein (Rho GTPase binding) 4 (CDC42EP4). In addition to being quite a mouthful, this protein resides in the cytoplasm and is associated with the actin filaments. In this image, CDC42EP4 is seen in U-2 OS human osteosarcoma cells.

As the name suggests, the CDC42EP4 is a protein associated with CDC42, which helps regulate the transition from G1 to S (in which DNA is replicated), and is essential for proper cell cycle progression (Yasuda S et al. 2006)...Read more


Systematic antibody validation using siRNA

2016-02-02
Cell Atlas Gene editing Immunocytochemistry Immunofluorescence siRNA Subcell Atlas

Fig 1a. The protein HNRNPUL1 is localized to the nucleus by staining with antibody HPA046290 (green)

Systematic antibody validation with siRNA for the Human Protein Atlas

Antibodies are among the most frequently used tools for basic research and clinical assays. For antibodies used in therapy or diagnostics, there are well-defined and strict guidelines that must be complied with before approval for clinical assays. For research antibodies, such guidelines have not yet been developed, despite the importance of demonstrating that they are specific, selective, and yield reproducible results in the immunoassay for which they are to be used...Read more


Validation of antibodies using GFP-tagged target

2015-12-15
Cell Atlas Co-localization HPA14 Immunocytochemistry Immunofluorescence Subcell Atlas Validation

Antibody HPA37362 targeting BOD1L1 overlaps with GFP-tagged target protein and the localization is identical to that of endogenous protein in non-transfected control cells

Version 14 of The Human Protein Atlas includes a new type of validation of antibodies that are used for determining the subcellular localization of a protein.

A set of antibodies have been analyzed in transgenic cell lines expressing GFP-tagged target protein at near-endogenous levels to confirm that the antibodies are capable of binding the target protein. The approved antibodies are then used to determine the subcellular localization of endogenous protein in a selection of cell lines. A high validation score is assigned to those genes where the same location(s) are observed for both tagged protein and protein detected using labelled antibody in non-transfected cells...Read more


Increasing reliability by co-localization

2015-11-10
Cell Atlas Co-localization HPA14 Immunocytochemistry Immunofluorescence Subcell Atlas Validation

Subcellular localization obtained using IF (left) and GFP (FP, right) for the endoplasmic protein FKBP7 (top) and the FHL2 protein localized to focal adhesion sites (bottom).

Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells

The Human Protein Atlas applies antibodies for a variety of applications to map protein expression in different tissues and also at the subcellular level. Within the subcellular protein atlas, immunofluorescence (IF) is used to uncover the localization of proteins to different organelles. To ensure an accurate localization of each and very protein, the antibodies have to be specific to their target protein...Read more