To generate HeLa-Kyoto cells stably expressing biotinylated mTagBFP-tagged GCP2, cells were co-transduced with GCP2 and BirA lentivirus (Abella et al., 2016 (link)) followed by hygromycin and puromycin selection. Resistant cells expressing mTagBFP were sorted by FACS (fluorescent assisted cell sorter) and cultured independently in 96 well plates. The isolated single-cell colonies were screened for HA-BirA expression by immunofluorescence staining (primary antibody: mouse anti-HA (F-7, Santa Cruz Biotechnology); secondary antibody: goat anti-mouse-FITC (Sigma)) and then using high throughput imaging (High throughput screening facility, Francis Crick Institute). The localisation of GCP2-mTagBFP-BAP was confirmed by live-cell fluorescence imaging using a spinning disc confocal microscope based on a NikonTI-E frame with a 100x 1.49 N.A. Nikon objective lens (Cairn Research, Faversham, UK). mTagBFP expressing colonies were further tested by western blotting to confirm the expression of GCP2-mTagBFP-BAP and HA-BirA.
When producing large cell cultures for purification, three days before harvesting cells (using trypsination), D-biotin (Sigma Aldrich) was added to a final concentration of 50 μM. Cell pellets were stored at -80°C until further use.
When producing large cell cultures for purification, three days before harvesting cells (using trypsination), D-biotin (Sigma Aldrich) was added to a final concentration of 50 μM. Cell pellets were stored at -80°C until further use.