pET28b-mEGFP-DHHC20

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	Fluorescent proteins were expressed in BL21(DE3) Gold. Briefly, 2 liters of fresh 2x YT media with kanamycin were directly inoculated with bacterial colonies from overnight plates. The cultures were grown to an OD600 of 0.8 at 37°C. The temperature was then reduced to 25°C and IPTG added to a final concentration of 1 mM from a 1 M stock. Protein induction was carried out overnight.

Protocol tips
Fluorescent proteins were expressed in BL21(DE3) Gold. Briefly, 2 liters of fresh 2x YT media with kanamycin were directly inoculated with bacterial colonies from overnight plates. The cultures were grown to an OD600 of 0.8 at 37°C. The temperature was then reduced to 25°C and IPTG added to a final concentration of 1 mM from a 1 M stock. Protein induction was carried out overnight.

Publication protocol

Fluorescent proteins were expressed in BL21(DE3) Gold. Briefly, 2 liters of fresh 2x YT media with kanamycin were directly inoculated with bacterial colonies from overnight plates. The cultures were grown to an OD600 of 0.8 at 37°C. The temperature was then reduced to 25°C and IPTG added to a final concentration of 1 mM from a 1 M stock. Protein induction was carried out overnight. Cells were harvested by centrifugation next morning and the colored pellets re-suspended in 100 ml of 40 mM TrisHCl, pH 7.4, 270 mM NaCl, 10 mM 2-ME, 10 mM MgCl2, DNase (Worthington Biochemicals), lysozyme, 1 mM PMSF, AEBSF, and 5mM BenzamidineHCl. Cells were lysed by sonication on ice. Cell debris was pelleted by centrifugation at 38,000g for 30 min at 4°C. The supernatant was incubated with 2 ml NiNTA HisPur resin for an hr at 4°C on a rotator. The column was poured by gravity and the resin thoroughly washed with 20 ml of 40 mM TrisHCl, pH 7.4, 270 mM NaCl, 10 mM 2-ME containing 10 mM imidazole and then with same buffer containing 50 mM imidazole. Proteins were eluted with 10 ml of 20 mM TrisHCl, pH7.4, 135 mM NaCl, 5 mM 2-ME containing 350 mM imidazole. The eluates were dialyzed against 2 liters of the same buffer without the imidazole overnight at 4°C. The proteins were then flash frozen in liquid nitrogen and stored in −80°C freezer. At time of use, the proteins were thawed overnight in a 4°C fridge, diluted 10- to 20 times and their concentrations determined using a 660nm protein assay kit with BSA standards (Thermo Scientific Pierce). The protein concentrations were converted to μM using calculated molar masses of 29330, 29270, 29436, and 29160 g/mol for mVenus, mCerulean, mEGFP, and mCherry respectively.

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