Purification and Characterization of GCaMP6m

The coding region of GCaMP6m was moved into a constitutive bacterial expression vector (pCP, generous gift from Nathan Shaner), using ligation-independent cloning [49 (link)]. E. coli colonies expressing GCaMP6m were picked for presence of 470 nm excited fluorescence and grown at 34°C for 48 hours in Circlegrow (MP Biomedicals) with Ampicillin. Bacterial pellets were lysed using BugBuster (Novagen) and Benzonase (Novagen). Cleared lysates were then His-tag purified using Protino Ni-TED 2000 packed columns (Macherey-Nagel). Purified fluorescent proteins were eluted in 1x PBS with imidazole pH 8 buffer solution. The protein was then concentrated and buffer exchanged into a pH 7.2 MOPS buffer using Vivaspin Turbo 15 (Sartorius) polyethersulfone ultrafiltration columns. The same buffer exchange columns were used to make all Ca²⁺-free and Ca²⁺-saturated samples for protein measurements, except for the Ca²⁺ titration experiments. All purified GCaMP6m Ca²⁺-free protein measurements for this work were carried out in 10 mM EGTA /100 mM KCl / 30 mM MOPS at pH 7.2. All purified GCaMP6m Ca²⁺-saturated protein measurements for this work were carried out in 10 mM CaEGTA /100 mM KCl /30 mM MOPS at pH 7.2.