Snap vista green

For confirming successful labeling of the Cas9-SNAP proteins with the BG-coupled oligonucleotides, BG-coupled and uncoupled oligonucleotides were mixed with either SpCas9-SNAP, SadCas9-SNAP or only the Cas9-SNAP proteins alone, reactions were incubated for one hour at 30°C. For the SNAP-Vista Green (NEB) substrate, the protein was incubated for 30 min on 30°C in the dark. After incubation, reactions (300 ng) were loaded on 6% SDS-PAGE gel and run at 80V for 160 min. Gels that were containing BG-Vista Green (NEB, SNAP-Vista Green), were imaged prior to silver staining. The green fluorescence signal of the SNAP-tag was detected with a UV transilluminator (Biorad). Subsequently, silver staining was completed using the Pierce Silver Stain Kit (Thermo Scientific) according to manufacturer instructions, and imaged with a UV transilluminator (Biorad).

All DNA restriction and modification enzymes and the fluorescent substrate for the OGT activity (SNAP-Vista Green™, hereinafter BG-FL) were purchased from New England Biolabs (Ipswich, MA); molecular biology kits for the plasmid preparations and DNA gel extractions (NucleoSpin^® Gel and PCR Clean-up^®) were from Macherey-Nagel GmbH (Germany); Lyophilised Thrombin Protease from GE Healthcare (Illinois, US). Eurofins Genomics (Germany) performed the oligonucleotides synthesis and the DNA sequencing service.

Whole overnight inducted E. coli BL21(DE3) cells were collected and the expression of the H⁵-derived fusion proteins was analysed by an in vitro assay with the fluorescent SNAP-Vista Green™ substrate (New England Biolabs, Ipswich, MA; hereinafter BG-FL), as previously described⁵⁸ (link)^,64 . The in vivo imaging was carried out as described by Merlo et al.⁵⁸ (link). Briefly, bacterial cells expressing the H⁵-SspCA onto cell surface were washed twice in PBS 1× and resuspended in 50.0 μl of the same buffer supplemented with 5.0 μM of the BG-FL. After incubation at 37.0 °C for 30.0 min, cells were washed twice, resuspended, and again incubated for 30.0 min at 37.0 °C, to allow the external diffusion of the unreacted substrate. Images were collected using a DM6 fluorescence microscope and Hamamatsu camera under the control of Leica LAS AF 6000 software; excitation and emission wavelengths used suitably for AlexaFluor488 dye were λ_ex = 490 nm; λ_em = 525 nm, respectively.