Prestige Antibodies® as Organelle Markers for Sub–cellular Localization Studies

Introduction

Many characterization studies of proteins require the localization of specific cellular structures such as organelles. Co-localization with organelle-specific antibodies corroborates sub-cellular location of the target protein and may thereby reveal the potential involvement of the protein in cellular processes.

Among the most highly characterized antibodies in industry are the Prestige Antibodies powered by Atlas Antibodies. Several Prestige Antibodies have been shown to work as defined organelle markers. Each Prestige Antibody has been uniquely tested and comes with over 700 images displaying expression and localization of proteins in a large variety of normal human tissues, cancer cells and cell lines. All images are publicly available via the Human Protein Atlas (www.proteinatlas.org), a web portal governed by the Human Protein Atlas (HPA) program.

The HPA program systematically explores the human proteome by combining highly characterized antibodies with massive protein profiling in human tissues and cells. Although this multi-disciplinary research program operates using a large-scale high-throughput approach, well-validated methods and applications such as immunohistochemistry (IHC) and immunofluorescence (IF) staining are used to ensure the high quality of these antibodies. The primary reagents generated from the HPA program are affinity-purified polyclonal antibodies raised towards recombinant Protein Epitope Signature Tag (PrEST) antigens.

All annotated images from the applications tested are made publicly available via the Human Protein Atlas (www.proteinatlas.org), where more than two thirds of the characterized antibodies are Prestige Antibodies .

Immunofluorescence and Confocal Microscopy

In order to acquire information of organelle localization and distribution into sub-cellular compartments, the performance of several anti-human protein-affinity reagents is tested in IF. Furthermore, the valuable features of confocal microscopy as an optical imaging technique are used to create high-resolution images of the many sub-structures in the cell. Combining the two offers a powerful tool for even deeper studies of the human proteome.

The HPA program has carefully selected three different human cell lines, A-431 epidermoid carcinoma, U-251 MG glioblastoma and U-205 osteosarcoma, for organelle mapping of the proteome. As Prestige Antibodies are studied by IF staining, three well-characterized organelle markers for nuclei, microtubules and endoplasmic reticulum are used correspondingly as specific probes. The high-resolution confocal images are annotated based on sub-cellular localization. In addition, staining intensity and characteristics are also part of an overall assessment as well as comparison to literature (if available). All these factors contribute to a final reliability score publicly available on line.

Among the Prestige Antibodies tested in IF, a set of specific antibodies is classified as organelle markers. These probes have been subjected to the well-established quality control by the HPA program, and are thus very well suited for sub-cellular localization studies by IF.

Learn more about Prestige Antibodies as Organelle Markers at sigma.com/organellemarkers