Computational protocol: On the regulation, function, and localization of the DNA-dependent ATPase PICH

Similar protocols

Protocol publication

[…] To monitor the ATPase activity of PICH, the following reaction solution was prepared: 25 mM triethanolamine pH 7.5, 13 mM Mg-acetate, 1.8 mM dithiothreitol, 5 mM ATP, 100 μg/ml bovine serum albumin, 3 mM phosphoenolpyruvate, 20 U/ml pyruvate kinase, and 20 U/ml lactic dehydrogenase (Sigma-Aldrich, St. Louise, MO, USA) (modified from Huang and Hackney ). To assay the ATPase activity of PICH, the protein was provided as an immunoprecipitate, linked to protein A agarose (Bio-Rad, Hercules, CA, USA), and analyzed in a total reaction volume of 125 μl, using a Ultrospec 3100 pro (GE Healthcare, Waukesha, WI, USA). ATPase activity was measured by monitoring the decrease of OD 340 nm, indicating conversion of NADH to NAD+. To this end, an initial OD 340 nm was set to 2.5–3.0 by addition of NADH (20 mg/ml), and samples were analyzed every 5 min for a total reaction time of 1 h. To obtain optimal results, the reaction mixture was gently stirred by pipetting up and down every 5 min. Graphical and statistical analyses were performed using SigmaPlot 11 (Systat Software Inc., Chicago, IL, USA). [...] HeLaS3 cells stably expressing histone 2B-GFP (Sillje et al. ) or histone 2B-mCherry (Steigemann et al. ) were seeded in eight-well chambers from Ibidi (Martinsried, Germany), treated as indicated and imaged using a Nikon ECLIPSE Ti microscope equipped with a CoolLED pE-1 excitation system and a ×20/0.75 air Plan Apo objective (Nikon, Tokyo, Japan). Images were acquired at multiple positions every 3 min. Following antibody microinjections, GFP fluorescence and DIC images were acquired at each time point with 20- and 2-ms exposure times, respectively. TexasRed fluorescence was imaged every fifth time point with a 40-ms exposure. For siRNA knockdown and rescue experiments, histone 2B-mCherry fluorescence was acquired at every time point with a 20-ms exposure time. DIC images were collected as described above. To collect and process data, MetaMorph 7.7 software (MDS Analytical Technologies, Sunnyvale, CA, USA) in combination with SigmaPlot 11 (Systat Software Inc., Chicago, IL, USA) was used. […]

Pipeline specifications

Software tools SigmaPlot, MetaMorph
Application Miscellaneous