Suprasil quartz spectroscopy cells

Diguanylate cyclase activity of purified recombinant TdcA (His₆-MBP-TdcA or His₆-NusA-TdcA) and recombinant WspR (His₆-WspR) was measured using an EnzChek® Pyrophosphate Assay (Invitrogen) with modifications⁷⁰ also summarized here. High-purity guanosine 5ʹ-triphosphate (GTP) was purchased from Affymetrix. Yeast pyrophophatase was purchased from Roche. A standard curve was produced for each temperature using 50, 75, 100, and 125 μM pyrophosphate. We used 0.5 mM GTP for all measurements of diguanylate cyclase activity. Data were collected using a Lambda 35 UV/VIS Spectrometer with a Peltier device (Perkin-Elmer). Measurements were done using SUPRASIL® quartz spectroscopy cells with a light path of 10 mm (Perkin-Elmer).

Purified His₆-MBP-TdcA and His₆-MBP-DosP were diluted in protein buffer C to a concentration of 1 mg/ml for His₆-MBP-TdcA and 1.15 mg/ml for His₆-MBP-DosP, which was an empirically standardization based on the A₂₈₀ peak of each sample. The absorbance spectra for His₆-MBP-TdcA and His₆-MBP-DosP were collected in the range of 260–700 nm using a Lambda 35 UV/VIS Spectrometer with a Peltier device with SUPRASIL® quartz spectroscopy cells with a light path of 10 mm (Perkin-Elmer). The spectral scan was repeated six times for His₆-MBP-TdcA and His₆-MBP-DosP and each measurement was baseline corrected for buffer absorbance. Absorbance of hemin and flavin mononucleotide (FMN) standards were measured in an identical manner except that these compounds were prepared in ethanol.