Fluorescent Labeling of SNAP-Tagged Proteins

Labelling of SNAP-YuaG or free SNAP for microscopy was performed as described before [17 (link)]. Briefly, for labelling with the cell permeable SNAP-Cell TMR-Star (NEB) (excitation maxima: 554 nm; emission maxima: 580 nm) or the cell impermeable SNAP-Surface 488 (NEB) (excitation maxima: 506 nm; emission maxima: 526 nm) 1 µl of the stock solution was added to 400 µl culture and incubated for 30 minutes at 37°C. Cells were washed twice with fresh CH media,incubated for 15 minutes at 37°C and analysed microscopically.
Microscopy was performed as described in Bach et al. 2014 [32 (link)]. Images were taken on Zeiss AxioImager M1 equipped with a Zeiss AxioCam HRm camera. An EC Plan-Neofluar 100x/1.30 Oil Ph3 objective was used. Digital images were acquired with the AxioVision (Zeiss) software and analysed using the AxioVision 4.6 software (Zeiss). Final image preparation was done in Adobe Photoshop CS2 (Adobe Systems Incorporated).