Calmodulin-neuronal Nitric Oxide Synthase fluorescence decay files

Description

Raw binary data files (*.sdt) were converted to ascii fluorescence decay files (*.am) and ascii instrument response function files (*.ai). Settings for maximum entropy fits are contained in *.go files.

Steps to reproduce

Laser pulses at 800 nm were generated by a cavity-dumped Coherent Mira Ti:sapphire laser at a repetition rate of ~3.7 MHz and focused into a photonic crystal fiber (Thorlabs NL-PM-750) for continuum generation. The output was passed through an interference filter (Chroma E480/40) and focused into the sample. Alternatively, laser pulses at 980 nm were frequency-doubled in a harmonic generator (Inrad). The instrument response function was recorded with scattering at 490 nm selected by a monochromator (Sciencetech 9030) with an 8-nm bandwidth from a sample of colloidal silica (Ludox, Aldrich) (0.02% v/v). Solutions of CaM-T34C-AF488 (with or without additional mutations, as specified) of 100 to 200 nM in high-Ca2+ sample buffer consisting of 100 mM KCl, 10 mM CaCl2, 2 mM EGTA, 1 mg/ml BSA, 50 mM Tris, pH 7.4 were mixed with a two to four-fold excess of nNOS (wild-type or with mutations specified). Fluorescence from Alexa Fluor 488 was selected at 520 nm by the monochromator with an 8 nm bandwidth and detected by a microchannel plate photomultiplier tube (Hamamatsu R3809U-50). Signals were processed by a TCSPC module (Becker & Hickl SPC-830).

Institutions

• University of Kansas

  KS, Lawrence

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