isTrap column in buffer A complemented with 50 mM imidazole. Protein was eluted with a single 250 mM imidazole step, and fractions were dialyzed overnight at 4uC against 25 mM HEPES pH 7.5, 10 mM NaCl. ECAM-containing fractions were subsequently loaded onto a Mono Q column equilibrated in the dialysis buffer and eluted with a linear gradient to 0.5 M NaCl. ECAM was further purified by gel filtration chromatography using a Superdex 200 column equilibrated in buffer A. The central fractions of the peak corresponding to ECAM were pooled and concentrated to 10 mg/mL. Preparation of activated forms of ECAM Methylamine activation experiments were performed by incubating pure ECAM with 200 mM methylamine hydrochloride and 200 mM Tris-HCl pH 8.0 for 2 h at 25uC and subsequently subjecting the sample to gel filtration chromatography as described above. The central fractions of the peak were pooled and concentrated to 10 mg/mL. The reactions ” with elastase and chymotrypsin were carried out using the same protocol. A 1:1 molar ratio of protease:ECAM was Structural Studies of a Bacterial a2-Macroglobulin incubated at 37uC for 10 minutes, and the reaction was stopped with 1 mM PMSF, and subsequently injected on a gel filtration column; only the central fractions of the peak were used for further experiments. Small-angle X-ray Scattering Measurements were recorded at the ID14-3 beamline of the European Synchrotron Radiation Facility. Prior to data collection a scattering curve of bovine serum albumin 8 Structural Studies of a Bacterial a2-Macroglobulin reference solution was recorded. Experiments were performed at concentrations of 8.0 mg/mL for native ECAM, 6.4 mg/mL for the methylamine-activated form, 7.6 mg/mL for elastase-incubated ECAM and 6.1 mg/mL for the chymotrypsinreacted ECAM. Between measurements, scattering from a buffer sample was recorded, and these data were subsequently subtracted from the respective sample curves. No radiation damage was observed during the ten 10 second exposure frames and all data were recorded at 25uC. Data were treated following default parameters of the PRIMUS software package. The radius of gyration Rg and the forward scattering value I were estimated using the Guinier approximation. Both parameters, as well the maximum particle dimension Dmax, were also calculated by the 
GNOM software. Ab initio models of ECAM were generated using GASBOR. A final average model was generated from 15 independent models using DAMAVER through their pairwise superposition. Electron microscopy – Activated ECAM at a concentration of 0.05 mg/mL was first applied to the clean side of carbon on mica and negatively stained with 1% sodium silico tungstate. A grid was placed on top of the carbon film, and subsequently air-dried. Images were taken under low dose conditions with a Polara microscope operating at 300 kV and a nominal magnification of 59,0006with a Gatan CCD HC-067047 biological activity camera . A total of 51,700 particles were selected using a semiautomatic particle selection procedure with the EMAN boxer routine and extracted into 1006100 pixel boxes. The images were CTF-corrected with CTFFIND3 and Bsoft and low-path-filtered to 25 and 10 A. Due to the inherent flexibility of ECAM, only 15,000 particles were used to calculate the final 3D ” model, since the inclusion of a larger number of particles decreased the resolution. reconstruction. The result is show in Fig. S3. Rows labeled 1 show the re-projections of our reconstruction whereas rows labeled 2 sho
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