- 标题
- 摘要
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- 实验方案
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RNA-protein UV-crosslinking Assay
摘要： RNA-protein interactions play a crucial role in every aspect of RNA metabolism, and also plays a major role in post-transcriptional gene regulation. RNA-binding proteins have been implicated in viral gene expression (Ray and Das, 2002) and microRNA-mediated gene regulation (Poria et al., 2016). Here we have described the protocol which (1) covalently links transiently interacting RNA-protein complexes by UV crosslinking, (2) removes the unprotected RNA by RNase digestion and (3) detects the RNA-protein complexes by SDS-PAGE analysis. This protocol provides a rapid and reliable means to directly assay RNA-protein interactions and their kinetics using purified proteins and also help in identifying novel RNA-protein interactions
关键词： UV-crosslinking,RNA-protein interaction,RNA-binding proteins
更新于2025-09-04 15:30:14
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[Springer Series on Chemical Sensors and Biosensors] Label-Free Biosensing Volume 16 (Advanced Materials, Devices and Applications) || Computational Modeling of Biomolecule Sensing with a Solid-State Membrane
摘要： In this work, we theoretically study the interaction between a solid-state membrane equipped with a nanopore and a tethered, negatively charged polymer chain subjected to a time-dependent applied electrolyte bias. In order to describe the movement of the chain in the biomolecule-membrane system immersed in an electrolyte solution, Brownian dynamics is used. We show that we can control the polymer’s equilibrium position with various applied electrolyte biases: for a sufficiently positive bias, the chain extends inside the pore, and the removal of the bias causes the polymer to leave the pore. Corresponding to a driven process, we find that the time it takes for a biomolecular chain to enter and extend into a nanopore in a positive bias almost increases linearly with chain length while the time it takes for a polymer chain to escape the nanopore is mainly governed by diffusion. In addition to attaching the polymer chain to the mouth of the nanopore, the chain is attached to a molecule with a radius larger than that of the nanopore’s, acting as a molecular stop. This allows the polymer to thread the nanopore but not translocate it. In this new system, the chain’s variation of movement was compared to that of the freely translocating polymer chain. The results show the free polymer having greater variation in the radial direction, indicating the restrictions imposed by the molecular stop and bias aid in controlling the position and movement of the polymer chain in the nanopore.
关键词： Solid-state membrane,Nanopore,Protein,Polymer,Brownian dynamics
更新于2025-09-04 15:30:14
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Salt-Dependent Rheology and Surface Tension of Protein Condensates Using Optical Traps
摘要： An increasing number of proteins with intrinsically disordered domains have been shown to phase separate in buffer to form liquidlike phases. These protein condensates serve as simple models for the investigation of the more complex membraneless organelles in cells. To understand the function of such proteins in cells, the material properties of the condensates they form are important. However, these material properties are not well understood. Here, we develop a novel method based on optical traps to study the frequency-dependent rheology and the surface tension of P-granule protein PGL-3 condensates as a function of salt concentration. We find that PGL-3 droplets are predominantly viscous but also exhibit elastic properties. As the salt concentration is reduced, their elastic modulus, viscosity, and surface tension increase. Our findings show that salt concentration has a strong influence on the rheology and dynamics of protein condensates suggesting an important role of electrostatic interactions for their material properties.
关键词： surface tension,rheology,protein condensates,salt concentration,optical traps
更新于2025-09-04 15:30:14