Hairpin Unfolding Assay
Hairpin Unfolding Assay - Related Videos
01:37
The Unfolded Protein Response
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2023
The ER is the hub of protein synthesis in a cell. It has robust systems to quality control protein folding and also for degradation of terminally misfolded proteins. Under normal conditions, a small proportion of misfolded proteins that cannot be salvaged need to be transported to the cytoplasm by the ER-associated degradation or ERAD pathways. However, if the ERAD cannot handle the misfolded proteins, the cell activates the unfolded protein response or UPR to adjust the protein folding...
02:38
Assessing Bacterial Chaperone Activity via Thermal Unfolding of a Model Protein
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2025
Source: Dahl, J. et al. Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo. J. Vis. Exp. (2016)This video demonstrates a fluorescence spectrophotometer-based assay to evaluate chaperone activity on a substrate protein under acid and heat stress. It outlines the steps for monitoring substrate unfolding and aggregation through light scattering, comparing conditions with and without the chaperone.
03:25
Recording Neural Activity of Unfolded Hippocampal Tissue Using Penetrating Microelectrode Array
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2025
This video demonstrates the procedure to set up a recording chamber with a penetrating microelectrode array (PMEA) to capture neural signals from an unfolded hippocampus. This method allows us to study the speed and direction of propagation of neural activity within the unfolded hippocampus in vitro.
09:48
Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array
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Cited by 2 •
2015
We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.
01:31
Regulation of the Unfolded Protein Response
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2023
Inositol-requiring kinase one or IRE1 is the most conserved eukaryotic unfolded protein response (UPR) receptor. It is a type I transmembrane protein kinase receptor with a distinctive site-specific RNase activity. As the binding mechanics of the misfolded proteins with the N-terminal domain of IRE-1 are unclear, three binding models — direct, indirect, and allosteric -- are proposed for receptor activation. Nevertheless, it is known that once a misfolded protein associates with IRE1, it...
FAQs
The assay’s optical readout depends on label separation. In the folded hairpin, the fluorophore and quencher occupy a defined arrangement; when the stem-loop opens, that arrangement changes and the fluorescence signal changes accordingly. This converts a structural transition that cannot be observed directly into a measurable output, allowing opening to be compared across molecular interaction or activity conditions.
Strand invasion, helicase action, nuclease activity, and applied force can all produce the same observable endpoint, but they represent different ways of perturbing the hairpin. Comparing fluorescence responses under these distinct triggers helps investigators connect opening with a particular molecular interaction or activity, rather than treating every signal change as equivalent.
Force-driven opening and enzyme-driven opening provide different experimental information. Applied force probes how the hairpin responds to a mechanical input, while helicase or nuclease activity connects the signal to an enzyme-associated process. Keeping these triggers conceptually separate helps researchers interpret fluorescence changes in terms of the physical or biochemical event that produced them.
A workflow begins with a labeled stem-loop containing paired complementary sequences. The prepared hairpin is then examined under a condition that may promote strand invasion, enzyme-dependent opening, or force-induced unfolding. Fluorescence is measured as the structure changes, and the resulting signal is interpreted as evidence of hairpin opening under that condition.
The fluorescence output can be used to compare conditions that differ in molecular interaction or enzyme activity. Different signal changes indicate different structural responses of the hairpin, although interpretation depends on which trigger was applied. This makes the assay useful for quantifying interactions and examining activity within a controlled nucleic-acid system.
In immunology and infection research, the assay can be directed toward pathogen and host nucleic-acid enzymes. It helps characterize how those enzymes interact with nucleic-acid structures and supports studies of how infectious agents manipulate them. The same fluorescence-based framework can also contribute to assay development by providing a measurable output for comparing molecular activities.