6His-mCherry–tagged STIM1 (residues 342 to 469) and single-cysteine 6His-STIM1 (residues 340 to 685, C437S, S512C) were expressed in Hi5 insect cells with the Bac-to-Bac Baculovirus Expression System (Thermo Fisher Scientific). The cells were lysed by sonication in buffer containing 20 mM Hepes and 300 mM NaCl (pH 7.5) (buffer A), and the supernatant was collected after centrifugation at 12,000g for 45 min. The supernatant was incubated with nickel–nitrilotriacetic acid beads (Qiagen) for 1 hour. The beads were washed with buffer A mixed with 50 mM imidazole, and the sample was eluted with buffer A mixed with 300 mM imidazole. The 6His-mCherry-STIM1 was then desalted with a column packed with Sephadex G-50 beads (Sigma-Aldrich) to remove imidazole.

The 6His-STIM1 was incubated with tobacco etch virus (TEV) protease overnight to cleave the 6His tag and then dialyzed into buffer containing 50 mM tris and 200 mM NaCl (pH 7.8) (buffer B). Ion exchange chromatography (HiTrap Q column, GE Healthcare) was used to obtain high purity of the protein sample. The protein was mixed with Alexa Fluor 647 C2 Maleimide dyes (Thermo Fisher Scientific) for more than 2 hours, and the free dyes were separated using a column packed with Sephadex G-50 beads (Sigma-Aldrich).

The net protein charges were positive for 6His-mCherry–tagged STIM1 that contains 51 negatively charged residues and 54 positively charged residues. Alexa Fluor 647–labeled STIM contains 42 negatively charged residues and 42 positively charged residues, which is charge neutral.

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