Abstract
Multiplexing of GFP based and immunofluorescence translocation assays enables easy acquisition of multiple readouts from the same cell in a single assay run. Immunofluorescence assays monitor translocation, phosphorylation, and up/down regulation of endogenous proteins. GFP-based assays monitor translocation of stably expressed GFP-fusion proteins. Such assays may be multiplexed along (vertical), across (horizontal), and between (branch) signal pathways. Examples of these strategies are presented: 1) The MK2-GFP assay monitors translocation of MK2-GFP from the nucleus to the cytoplasm in response to stimulation of the p38 pathway. By applying different immunofluorescent assays to the MK2 assay, a multiplexed HCA system is created for deconvolution of p38 pathway activation including assay readouts for MK2, p38, NFκB, and c-Jun. 2) A method for evaluating GPCR activation and internalization in a single assay run has been established by multiplexing GFP-based internalization assays with immunofluorescence assays for downstream transducers of GPCR activity: pCREB (cAMP sensor), NFATc1 (Ca2+ sensor), and ERK (G-protein activation). Activation of the AT1 receptor is given as an example. 3) Cell toxicity readouts can be linked to primary readouts of interest via acquisition of secondary parameters describing cellular morphology. This approach is used to flag cytotoxic compounds and deselect false positives. The ATF6 Redistribution assay is provided as an example. These multiplex strategies provide a unique opportunity to enhance HCA data quality and save time during drug discovery. From a single assay run, several assay readouts are obtained that help the user to deconvolute the mode of action of test compounds.
Keywords: Multiplex, redistribution, GFP, high content, cell-based assay, p38, GPCR, cytotoxicity
Combinatorial Chemistry & High Throughput Screening
Title: Profiling of Multiple Signal Pathway Activities by Multiplexing Antibody and GFP-Based Translocation Assays
Volume: 11 Issue: 7
Author(s): Ulla Henriksen, Jacob Fog, Frosty Loechel and Morten Praestegaard
Affiliation:
Keywords: Multiplex, redistribution, GFP, high content, cell-based assay, p38, GPCR, cytotoxicity
Abstract: Multiplexing of GFP based and immunofluorescence translocation assays enables easy acquisition of multiple readouts from the same cell in a single assay run. Immunofluorescence assays monitor translocation, phosphorylation, and up/down regulation of endogenous proteins. GFP-based assays monitor translocation of stably expressed GFP-fusion proteins. Such assays may be multiplexed along (vertical), across (horizontal), and between (branch) signal pathways. Examples of these strategies are presented: 1) The MK2-GFP assay monitors translocation of MK2-GFP from the nucleus to the cytoplasm in response to stimulation of the p38 pathway. By applying different immunofluorescent assays to the MK2 assay, a multiplexed HCA system is created for deconvolution of p38 pathway activation including assay readouts for MK2, p38, NFκB, and c-Jun. 2) A method for evaluating GPCR activation and internalization in a single assay run has been established by multiplexing GFP-based internalization assays with immunofluorescence assays for downstream transducers of GPCR activity: pCREB (cAMP sensor), NFATc1 (Ca2+ sensor), and ERK (G-protein activation). Activation of the AT1 receptor is given as an example. 3) Cell toxicity readouts can be linked to primary readouts of interest via acquisition of secondary parameters describing cellular morphology. This approach is used to flag cytotoxic compounds and deselect false positives. The ATF6 Redistribution assay is provided as an example. These multiplex strategies provide a unique opportunity to enhance HCA data quality and save time during drug discovery. From a single assay run, several assay readouts are obtained that help the user to deconvolute the mode of action of test compounds.
Export Options
About this article
Cite this article as:
Henriksen Ulla, Fog Jacob, Loechel Frosty and Praestegaard Morten, Profiling of Multiple Signal Pathway Activities by Multiplexing Antibody and GFP-Based Translocation Assays, Combinatorial Chemistry & High Throughput Screening 2008; 11 (7) . https://dx.doi.org/10.2174/138620708785204081
DOI https://dx.doi.org/10.2174/138620708785204081 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
Call for Papers in Thematic Issues
Artificial Intelligence Methods for Biomedical, Biochemical and Bioinformatics Problems
Recently, a large number of technologies based on artificial intelligence have been developed and applied to solve a diverse range of problems in the areas of biomedical, biochemical and bioinformatics problems. By utilizing powerful computing resources and massive amounts of data, methods based on artificial intelligence can significantly improve the ...read more
Eco-friendly Agents for Biological Control of Pathogenic Diseases
The discovery of an alternative biological approach to disease management includes work on medicinal products derived from natural sources as a starting point for the development of eco-friendly agents for these diseases and the injuries they cause, as well as reducing human contact with hazardous chemicals and their residues. We ...read more
Emerging trends in diseases mechanisms, noble drug targets and therapeutic strategies: focus on immunological and inflammatory disorders
Recently infectious and inflammatory diseases have been a key concern worldwide due to tremendous morbidity and mortality world Wide. Recent, nCOVID-9 pandemic is a good example for the emerging infectious disease outbreak. The world is facing many emerging and re-emerging diseases out breaks at present however, there is huge lack ...read more
Exploring Spectral Graph Theory in Combinatorial Chemistry
Scope of the Thematic Issue: Combinatorial chemistry involves the synthesis and analysis of a large number of diverse compounds simultaneously. Traditional methods rely on brute force experimentation, which can be time-consuming and resource-intensive. Spectral Graph Theory, a branch of mathematics dealing with the properties of graphs in relation to the ...read more
- Author Guidelines
- Graphical Abstracts
- Fabricating and Stating False Information
- Research Misconduct
- Post Publication Discussions and Corrections
- Publishing Ethics and Rectitude
- Increase Visibility of Your Article
- Archiving Policies
- Peer Review Workflow
- Order Your Article Before Print
- Promote Your Article
- Manuscript Transfer Facility
- Editorial Policies
- Allegations from Whistleblowers
Related Articles
-
Cervical Cancer Diagnosis: Insights into Biochemical Biomarkers and Imaging Techniques
Combinatorial Chemistry & High Throughput Screening GRP78 Influences Chemoresistance and Prognosis in Cancer
Current Drug Targets Heparan Sulfate Proteoglycans, Tumour Progression and the Cancer Stem Cell Niche
Current Cancer Therapy Reviews Identification of HIV Inhibitors Guided by Free Energy Perturbation Calculations
Current Pharmaceutical Design Application of Baculovirus-Insect Cell Expression System for Human Therapy
Current Pharmaceutical Biotechnology MicroRNAs as Cancer Biomarkers
MicroRNA Advances in Anticancer Antibody-Drug Conjugates and Immunotoxins
Recent Patents on Anti-Cancer Drug Discovery NF-κB-IKKβ Pathway as a Target for Drug Development: Realities, Challenges and Perspectives
Current Drug Targets Structure-based Virtual Screening for the Identification of High-affinity Small Molecule Towards STAT3 for the Clinical Treatment of Osteosarcoma
Current Topics in Medicinal Chemistry Novel Agents Targeting Bioactive Sphingolipids for the Treatment of Cancer
Current Medicinal Chemistry Curcumin: Structure-Activity Relationship Towards its Role as a Versatile Multi-Targeted Therapeutics
Mini-Reviews in Organic Chemistry Targeted Drug Delivery in Brain Tumors-nanochemistry Applications and Advances
Current Topics in Medicinal Chemistry Pharmacogenetics of Phase I and Phase II Drug Metabolism
Current Pharmaceutical Design Enhancing the Cytotoxic Activity of Novel Targeted Therapies – Is There a Role for a Combinatorial Approach?
Current Clinical Pharmacology MicroRNA-34 Family, Mechanisms of Action in Cancer: A Review
Current Cancer Drug Targets Regulators of Chemokine Receptor Activity as Promising Anticancer Therapeutics
Current Cancer Drug Targets Editorial: (Thematic Issue: Nanofluidics and Microfluidics: Novel Approaches in Biomedical Science)
Current Proteomics Selective Inhibitors of Zinc-Dependent Histone Deacetylases. Therapeutic Targets Relevant to Cancer
Current Pharmaceutical Design Oxidative Stress, Pro-Inflammatory Cytokines, and Antioxidants Regulate Expression Levels of MicroRNAs in Parkinson’s Disease
Current Aging Science Augmenting T Helper Cell Immunity in Cancer
Current Drug Targets - Immune, Endocrine & Metabolic Disorders