Naturally occurring toxins have been invaluable tools for the study of structural and functional relationships of voltage-gated sodium channels (VGSC). at 37?C. After an induction period of 3?h, the cells were harvested by centrifugation and washed with 20?mM Tris-HCl, pH 8.0. The cell pellet from 4?liters of buy 29477-83-6 culture was suspended in buy 29477-83-6 100?mL of 20?mM Tris-HCl, pH 8.0, 0.2?mM phenylmethylsulfonyl fluoride, and the cells were lysed using a probe sonicator. The lysate was centrifuged at 30,000??g Rabbit polyclonal to Tumstatin. and 10?C for 30?min and the supernatant was dialyzed against 20?mM phosphate buffer, pH 8.0 for 24?h. After centrifugation and filtration, the proteins were purified by cation exchange chromatography on SP-Sepharose and size exclusion chromatography on Sephacryl S-100. The protein was further purified by additional gel filtration on Superdex 75. A selenomethionine derivative of FS50 was obtained by growing B834(DE3)pLysS with the plasmid construct described above in SelenoMet media supplemented with selenomethionine (Molecular Dimensions) as per buy 29477-83-6 the manufacturers instructions. The protein was purified using the methods described above. Patch clamp recording on rat dorsal root ganglion (DRG) neurons Rat DRG neurons were acutely dissociated and maintained in a short-term primary culture according to procedures adapted from Xiao that blocks the sodium channel NaV1.5. Sci. buy 29477-83-6 Rep. 6, 36574; doi: 10.1038/srep36574 (2016). Publishers note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgments The authors thank the staff of the Southeast Regional Collaborative Access Team, Advanced Photon Source, Argonne National Laboratory for assistance with X-ray data collection. This work was supported in part by the Chinese National Natural Science Foundation (3137078), the Chinese Ministry of Science buy 29477-83-6 and Technology (2010CB529800) and the National Institutes of Health intramural research program of NIAID. Footnotes Author Contributions X.X. designed experiments, performed experiments and analyzed data; B.Z., S.A. and S.Y. performed experiments and analyzed data; J.F.A. designed experiments, performed experiments and analyzed data; X.X., J.M.C.R., B.Z., S.A., S.Y. and J.F.A. contributed to the manuscript..