Similar protocols

Pipeline publication

[…] , and the digest fragment F1 (0.35 μm) were recorded at 10 nm/min; data pitch, 0.1 nm; response time, 2 s between 185 and 285 nm in a 0.1-cm path length quartz cuvette at 25 °C (JASCO-810 spectrometer). The proteins were exchanged into 10 mm sodium phosphate, pH 7.6, 150 mm sodium fluoride prior to analysis (HiTrap desalt column, GE) at 4 ml/min. Spectra were corrected by subtracting a buffer baseline, each an average of 5 spectra. Secondary structure was estimated using the Dichroweb CD secondary structure analysis server () including the methods CONTIN, SELCON3, and CDSSTR () and reference data sets SP175 and 7 ()., The secondary structure of lymphostatin was predicted from sequence using PredictProtein () and PSIPRED (). Proteins with similar structural elements and homologues were identified with PHYRE () and BLASTp ()., Binding of uridine diphosphate-glucose (UDP-Glc) and UDP-GlcNAc to wild-type LifA and LifADTD/AAA was determined by ligand-induced changes in intrinsic tryptophan fluorescence. Fluorescence measurements were performed on a SPEX Fluoromax 3 spectrometer (Horiba) in a 3-ml stirred cuvette by titrating the UDP-sugar into 0.2 μm lymphostatin at 20 °C in 20 mm sodium phosphate, pH 7.6, 150 mm sodium chloride, 5% glycerol, 0.1% Tween 20, 1 mm DTT. Samples were allowed to equilibrate for 5 min after the addition of each aliquot. The final volume added did not exceed 2% of the initial volume. Tryptophan was excited at 295 nm and emission spectra were recorded from 310 to 400 nm, with a 1 […]

Pipeline specifications

Software tools PP, PSIPRED, Phyre, BLASTP