THE HUMAN PROTEIN ATLAS BLOG
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
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
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 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
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
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