Alexa fluor 568 protein labelling kit

The MakA protein was purified as previously reported [4 (link)]. For the immunofluorescence experiments, MakA was labelled with Alexa Fluor 568 using an Alexa Fluor 568 protein labelling kit (Thermo Fisher) according to the manufacturer’s instructions.

Recombinant proteins were diluted to the indicated salt concentrations with buffer containing 10 mM Tris-HCl pH 7.5 to induce phase separation. A total of 8 μl of phase-separation solution was loaded onto a glass slide, covered with a coverslip and images were acquired using the Zeiss LSM880 microscope. For identifying droplet fusion events, glass slides loaded with protein solutions were inverted on the microscope lens, and images were acquired at 1 s intervals and further analysed using ImageJ. For FRAP assays, droplets containing fluorescent proteins were bleached with the desired laser intensity and 100 post-bleach frames were recorded with a time interval of 1 s. The fluorescence intensity at bleached region was corrected with an unbleached region and normalized to the pre-bleaching fluorescence intensity. For the co-phase separation assay of wild-type or mutant GFP–SSB1 with INTAC, INTAC was labelled using the Alexa Fluor 568 protein labelling kit (Thermo Fisher scientific) according to manufacturer’s protocols. The labelled INTAC proteins were diluted with unlabelled ones to a desired concentration and then mixed with GFP fusion proteins to induce phase separation.