3 mM) (Tricine-SDS-PAGE) with tricine-containing cathode buffer as previously described [36]. Stacking and separating gels contained 5.5% and 10% (v/v) acrylamide, respectively. Following the electrophoresis of LOS samples, gels were fixed and the resolved molecules were detected using the carbohydrate silver staining method [37] or CPS by Alcian Blue staining [38]. Electrophoresis was conducted at 30 V for 1 h to maximize stacking and then separated at 200 V for 30 min. Whole-cell

protein samples were resolved on glycine-buffered 15% (v/v) polyacrylamide gels (Glycine-SDS-PAGE) as previously described [39]. Electrophoresis was conducted at 100 V for 1.5 h. Proteins were detected by conventional Coomassie Repotrectinib manufacturer Blue staining

[19]. Densitometry image analysis was performed using the QuantityOne selleck chemicals software package (Bio-Rad). The published M. catarrhalis LOS from M. catarrhalis wild-type (strain 2951) and the lgt4 LOS biosynthesis mutant [24] were used as a eFT508 ic50 control for relative size determination of LOS structures due to the loss of a single hexose sugar from the known OS structure. NMR spectroscopy Purified OSs were dissolved in D2O (CIL 99.998%) and cycled through 3 steps of lyophilization/dissolution to remove exchangeable protons. 1H and 13C NMR experiments were performed at 600 MHz and 150 MHz respectively at 298 K or 278 K in D2O using a Bruker Avance spectrometer. Chemical shifts are reported in ppm referenced to DSS. Spectral assignment was aided by recording of 1H 1D, gradient correlation spectroscopy (COSY), TOCSY, (60 and 120 ms mixing Adenylyl cyclase time), 13C attached proton test (APT), 1H-13C-HSQC

and edited 1H-13C-HSQC (CH and CH2 correlations opposite sign), 1H-13C-HSQC-TOCSY and edited 1H-13C-HSQC-TOCSY (60 and 120 ms mixing time) (one bond C-H correlations opposite sign), and 1H-13C-HSQC-nuclear Overhauser enhancer spectroscopy (-NOESY), NOESY (400 ms) spectra. In addition, 1D selective TOCSY experiments were used to assist with the assignment process. All spectra were acquired using unmodified pulse sequences from the Bruker pulse sequence library. Ligand and Western blotting In addition to chemical staining, the fractionated C. jejuni LOS was transferred from Tricine SDS-PAGE gels onto a Pall® PVDF membrane using a semi-dry transblotter (Bio-Rad). After transfer, the membrane was reacted with horseradish peroxidase-(HRP-) conjugated CTB (3 μg mL-1), or with HRP-conjugated PNA (lectin from Arachis hypogaea) (5 μg mL-1), or with HRP-conjugated anti-GM1 ganglioside IgG (diluted 1:3000) in PBS. Membranes were developed using HRP Color Development Solution (Bio-Rad) or SuperSignal HRP Chemiluminescent Substrate (Thermo Scientific) according to the manufacturer’s instructions. Colony lift C.