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AKTA pure chromatography system

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Heterologous Expression and Purification of SARS-CoV2 Nucleocapsid Protein
[Abstract]  This protocol describes a step by step method for heterologous expression of SARS-CoV2 Nucleocapsid (N) protein in Escherichia coli. Moreover, this protocol includes steps to purify the N protein to high purity and homogeneity. Thus, purified protein can be used for ligand binding assays and other biochemical experiments.

Preparation, FPLC Purification and LC-FT-ICR-MS of Proteins
[Abstract]  High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers provide extremely high mass resolution (resolving power of ~200,000 at 400 m/z) protein detection across a broad mass range, enabling analysis of fine structure of isotopic peak clusters that is missed in other types of mass spectrometers. The protocol detailed here describes preparation of cellular extracts for purification of DNA-binding proteins using multiple chromatographic chemistries via fast protein liquid chromatography (FPLC), and identification and quantitation of the protein isoforms and their post-translational modifications by liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FT-ICR-MS). This protocol benefits from selectively purifying ...

In vitro RNA Cleavage Assays to Characterize IRE1-dependent RNA Decay
[Abstract]  The kinase/RNase IRE1 is a key effector of the cellular response to endoplasmic reticulum stress. The RNase activity of IRE1 can be measured in cells or in the test tube. Here we describe a protocol for the in vitro cleavage and analysis of RNA substrates of IRE1. The method consists of the in vitro transcription, purification and re-folding of IRE1 substrate RNAs followed by their cleavage using recombinant cytosolic kinase/RNase domains of IRE1 and the separation of the resulting fragments by denaturing polyacrylamide gel electrophoresis. This protocol allows the study of the cleavage kinetics of IRE1’s RNA substrates in vitro.