Alexa fluor 488 protein labelling kit

The MTII protein used in these studies was native rabbit Zn₇-MT-IIA (the major brain-expressed mammalian MTII isoform) purified by high-performance liquid chromatography (Bestenbalt LLC, Estonia). The MTII was N-terminally tagged using the AlexaFluor® 488 Protein Labelling Kit (Invitrogen) in accordance with the accompanying protocols. During the column fractionation of labelled protein, all of the eluted fractions were collected including the unincorporated dye (the second eluted fluorescent peak). The fractions collected from the first eluted fluorescent peak contained the MTII conjugated with the AlexaFluor 488 tag and is hereafter referred to as MT₄₈₈, while the unincorporated dye peak was used as the vehicle treatment and is referred to as A₄₈₈.

Briefly, E. coli strain, BL21 (DE3), was separately transformed with plasmids encoding the proteins. Then, the cGAS proteins were expressed and purified as previously described^{1 (link)}. The E. coli cells were grown at 37 °C, until an OD₆₀₀ of 0.6 was reached. The temperature was then reduced to 20 °C, and the cells were induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 24 h at 20˚C. The resultant cells were harvested by centrifugation at 5000 x g for 15 min and washed twice with cold phosphate-buffered saline (PBS). The collected cells were broken by ultrasonic wave and centrifugated at 24,000 x g for 30 min to remove unbroken cells and debris. The soluble fraction was incubated by Ni Sepharose 6 Fast Flow (Cytiva), washed with binding buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 30 mM imidazole), and eluted with elution buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 300 mM imidazole) as previously described^{1 (link)}. Concentration of resultant proteins and buffer exchange were performed using an Amicon Ultrafree centrifugal filter (Millipore) with a cutoff of 10 kDa. The proteins were labelled with Alexa Fluor™ 488 using the Alexa Fluor™ 488 Protein Labelling Kit (ThermoFisher, Waltham, MA, USA). The estimated degree of labeling was 2 mol of Alexa Fluor 488 per mol of protein.