The molecular mass of ArsR protein ArsR1 or ArsR2 was measured using an ÄKTA Pure fast protein liquid chromatography system (Cytiva, Marlborough, MA, USA). First, 500 μL of 40 μM ArsR1 or 37 μM ArsR2 protein in 10 mM MOPS/0.1 M NaCl/15% glycerol buffer (pH 7.5) was loaded onto a Superose 12 column (Cytiva, Marlborough, MA, USA) pre-equilibrated by the same buffer at a flow rate 0.5 mL min−1 and elution was monitored at 280 nm. The calibration was carried out with gel filtration standards ribonuclease A, chymotrypsinogen A, ovalbumin, and albumin in a concentration of 1 mg mL−1. Then, 100 μM sodium arsenite or 1 mM diamide was mixed with the proteins and incubated for 10 min at 30 °C to investigate how these compounds influence ArsR oligomeric state.
Kta pure fast protein liquid chromatography system
Manufactured by Cytiva
Sourced in United States
The ÄKTA Pure is a fast protein liquid chromatography (FPLC) system designed for protein purification and analysis. It provides precise control and monitoring of parameters such as flow rate, pressure, and UV absorption during the chromatographic process. The system is intended for use in laboratories conducting research and development in the biopharmaceutical and life sciences industries.
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2 protocols using kta pure fast protein liquid chromatography system
1
ArsR Protein Oligomeric State Analysis
2
TIMP-1 and N-TIMP-1 Signaling in HEK293F Cells
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Recombinant human (N-)TIMP-1 was expressed in an endotoxin-free mammalian cell culture system of HEK293F cells (Thermo Fisher Scientific) and purified in a three-step protocol using an Äktapure Fast Protein Liquid Chromatography system (Cytiva) as described previously (16 (link)). For stimulation of cells for subsequent analysis of intracellular signaling, cells were incubated in serum-free media containing 0.1% BSA for 24 h before stimulation. After serum starvation, cells were exposed to pathophysiological concentrations of TIMP-1 (500 ng/ml) (14 (link)), N-TIMP-1 (392 ng/ml, equimolar to TIMP-1), or MIF (100 ng/ml, Abcam, cat. #ab75432) for 10 min and whole-cell extracts were isolated using RIPA lysis buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.25% SDS, 1% NP-40, 1 mM EDTA) supplemented with protease and phosphatase inhibitors (Carl Roth). Concentrations of isolated proteins were determined using the Pierce BCA Protein Assay Reagents (Thermo Fisher Scientific Inc). For interference with TIMP-1–CD74–mediated signaling, cells were preincubated with the CD74-blocking peptide C36L1 (37 (link)) (200 μM, KSSQSVFYSSNNKNYLA-NH2, peptides & elephants GmbH) for 6 h before stimulation.
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