Reconstitution of hTRPV6 protein into amphipols
This protocol is extracted from research article:
Mechanism of calmodulin inactivation of the calcium-selective TRP channel TRPV6
Sci Adv, Aug 15, 2018; DOI: 10.1126/sciadv.aau6088

hTRPV6 was incorporated into amphipols as described previously (29). Briefly, for reconstitution in A8-35 amphipols (Anatrace #A835), purified hTRPV6 in the presence of 20 μM CaM and 5 mM Ca2+ was mixed with amphipols in a 1:3 mass ratio (protein/amphipols) and incubated for 3 hours with rotation at 4°C. Subsequently, 7 to 8 mg (per 0.5 ml of protein/amphipol mixture) of Bio-beads SM2 (Bio-Rad) prewet in buffer containing 20 mM tris (pH 8.0), 150 mM NaCl, and 1 mM β-mercaptoethanol was added to the protein/amphipol mixture to facilitate the reconstitution of TRPV6 into amphipols. The mixture was rotated for ~20 hours at 4°C, and the amphipol-solubilized TRPV6 was purified as described above.

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