All You Need to Know about Nuclear Translocation Assays

All You Need to Know about Nuclear Translocation Assays

Upon stimulation, many proteins translocate into the nucleus to regulate various cellular processes. Protein translocation between cellular compartments is a critical process in cell biology; hence, this assay is applied in multiple biological and medical studies to understand disease mechanisms. This blog post will explain the basics of this assay and its contribution to various cell-based experiments.

What is Nuclear Translocation Assay?

Many cellular physiological processes, including cell division, cell differentiation, cell motility, immune response, neural transmission and apoptosis, are activated and regulated through Signal transduction pathways.

The activation of a signaling pathway usually results in the transport of an extracellular biological signal into a cell compartment (cytoplasm or nucleus), followed by a physiological response from the cell, E.g. activation of transcription factors which results in the expression of specific gene products. The translocation of molecules often generates the cell’s physiological response to a signal from one cellular compartment to another. These molecules include macromolecules, such as transcription factors, protein kinases, or smaller molecules (second messengers). To appropriately carry out critical cellular functions, cellular signal transduction pathways are highly complex and involve multiple regulatory mechanisms with significant crosstalk and redundancy. Such complex signaling pathways include the MAPK pathway, NFkB mediated pathway, PI3K mediated pathway and G protein pathway. These pathways have been indicated in many diseases, and understanding them has led to an immense interest in targeting them to treat certain conditions. 

The canonical regulation of transcription factors, such as c-fos, c-jun, NFkB and Kinases, such as MAP Kinase, is via the translocation of the transcription factor from the cytoplasm into the nucleus.

Transcription factor proteins enter the nucleus by being transported there through nuclear pores (often by a particular transport pathway).

Nuclear Translocation assay
Nuclear Translocation assay using DAPI + FAR RED labeling and 10X magnification Automated image analysis, performed simultaneously with acquisition, identifies wells demonstrating high nuclear translocation upon treatment (red), in comparison to the low translocation measured in control wells (blue).

How & Why Would you Visualize the Nuclear Translocation Process?

The nuclear translocation assay (NTA) is a novel approach for investigating intracellular protein-protein interactions. Few assays allow for investigating these interactions in the cellular setting in which they occur.

Combining Hermes high-content screening technology with a cell-based reporter gene assay offers the complete solution for a fruitful screening campaign for protein translocation from one cell compartment to another.

By visualizing the protein in the various compartments, measuring its intensity, and calculating the ratio of the signal intensity of a labeled protein between the cytoplasmic and nuclear compartments as a quantitative measure of translocation, imaging is a valuable method for monitoring and measuring the translocation process.

The cytoplasm to nuclear translocation assay by Athena image analysis software measures the activation and/or translocation of one object at a time at the single cell level. It analyzes images and measures ratios of fluorescence intensities in the nuclear and cytoplasmic regions on a single cell level.

Translocation assay
Protein translocation between Cytoplasm to Nucleus, captured with Hermes automated microscope
LPS-mediated activation of IRF3: Nuclear translocation of IRF3 Interferon regulatory factor 3 (red) in response to stimulation with LPS

Nuclear Translocation Assay & Spot Detection for Microbiology Studies

IDEA Bio-Medical offers a specialized nuclear translocation assay, dedicated to microbiology studies. This assay allows measuring and counting the number of intracellular bacteria or viral infections within a specific cell compartment on a cell-by-cell basis.

This application detects Single cells and divides intracellular spaces into nuclear and cytoplasmic compartments to allow compartment-specific measurement and count: granules, foci, or other spots within each cell.

What features does Nuclear Translocation Assay by IDEA Bio Offer?

  • High-throughput acquisition without compromising image quality
  • A unique statistical approach for robust measurements
  • Automatically detect each cell and nucleus and measure the fluorescence intensity ratio between these two compartments
  • Users can obtain quantitative results during image acquisition to save both time and labor

Automatically quantify nuclear translocation of proteins from one cell compartment to another with IDEA Bio-Medical.

References and further reading:

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