Bioorthogonal reactions, including the strain-promoted azideCalkyne cycloaddition (SPAAC) and inverse electron demand DielsCAlder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained isomerization. (W) … We next examined the comparative stability of the TCO series of HaloTag ligands (compounds 4C7) following conjugation to Halo-H2B-GFP in the nucleus or Halo-KDEL in the endoplasmic reticulum (ER) (Physique ?Physique44C,Deb). HeLa cells were transfected with either Halo-H2B-GFP or Halo-KDEL and HaloTag ligands 4C7 were added to make the corresponding clickable HaloTag conjugates and extra unbound ligand was washed out of the cells. TAMRA-Tz 13 was added at the indicated time points over 24 h to evaluate how much of the TCO-Halo conjugate was stable and could undergo the iEDDA reaction. Results were quantified by in-gel fluorescence and Western blotting. The reaction of TCO 4-Halo-H2B-GFP with TAMRA-Tz 13 progressed to the same extent over 24 h indicating that TCO 4 was stable in the nucleus under these conditions. In contrast, less TAMRA-Tz 13 reacted over the 24 h period with the conformationally strained TCO derivatives presumably due to isomerization to the unreactive for 3 min, the buffer was aspirated and cell pellets were immediately iced on dry ice. HeLa Cell Lysis, SDS-PAGE, In-Gel Fluorescence, and Western Blotting For SPAAC, cell pellets were lysed by sonication in 100 L 1% SDS/SPAAC quench buffer. Rabbit polyclonal to FOXRED2 For iEDDA, cell pellets were lysed by sonication in 100 L 1% SDS/iEDDA quench buffer. Protein concentrations were decided with a BCA protein assay (Thermo-Fisher) and cell lysates were normalized by protein concentration. Samples were prepared in 1 LDS sample buffer (Life Technologies), with 10 g protein loaded per well, and separated by SDS-PAGE on NuPage 4C12% Bis-Tris gels in MES running buffer (Life Technologies). TAMRA-fluorescence was analyzed on a Typhoon variable mode imager (GE Healthsciences) using a TAMRA filter. Gels were then transferred to nitrocellulose using iBLOT (Life Technologies), blocked in Odyssey blocking buffer (LiCor) for 1 h at RT, and incubated in anti-HaloTag pAb (Promega, G9281) at 1:2000 overnight in TBST. Membranes were washed 3 occasions in TBST, and incubated in goat anti-rabbit IRDye 800CW (LiCor) at 1:10,000 in TBST for 1 h at RT. Membranes were washed three occasions in TBST and imaged on the Odyssey Infrared Imager (LiCor). Data Quantification Fluorescence intensity measurements were quantified in ImageJ 1.45 (NIH) for both TAMRA fluorescence and total HaloTag protein expression. In-gel fluorescence (TAMRA) signal first was 63388-44-3 supplier normalized to total HaloTag protein manifestation signal (Western blot). The positive TAMRA-control (ligand 12) was set at 100% for each experiment. SPAAC and iEDDA data were normalized to this value, and reported as a percent of control. Data from 3 to 6 impartial replicates were quantified and plotted as the log[dose] vs response for generation of EC50 values. Curves were fit using a four parameter doseCresponse curve in GraphPad Prism version 6.03 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com. For timecourse experiments, data was plotted as the percent of control vs time and fit with pseudo-first order association kinetics where = = rate constant expressed as the reciprocal of in models. for 3 min, and processed as described previously for 63388-44-3 supplier in-gel fluorescence. 63388-44-3 supplier Each time point contained 12 as a control to account for labeled HaloTag protein degradation during the course of the experiment. Evaluation of SPAAC and iEDDA Reactions for Live/Fixed Cell Imaging HeLa cells were plated on poly-lysine coated glass-bottom dishes (MatTek, P35GC-1.5C14C), transfected, and treated as described for in-gel fluorescence with the following modifications. Following fluorophore incubation in culture media, cells were washed 3 1 mL PBS with the appropriate quench reagent, and quenched for an additional 5 min in media. Cells were washed 3 1 mL media to remove quench reagent and incubated in cell culture media for 1C2 h prior to imaging. Cells were washed one time in phenol red-free DMEM (Life Technologies)/10% FBS, and media was replaced with phenol-red free DMEM/10% FBS supplemented with 10 g/mL Hoescht 33342 (Life Technologies, H3570) for nuclear labeling 5 min prior to imaging live. For fixed-cell imaging, cells were fixed in ice-cold MeOH for 10 min, washed 3 1 mL PBS and incubated in 1 mL PBS overnight at 4 C. PBS was aspirated and 100 L VECTASHIELD (Vector Laboratories) cell mounting media made up of DAPI (Vector Laboratories, H-1200) was added prior to imaging. Fluorescence Microscopy Live and fixed cells were imaged on a Zeiss AxioObserver.Z1 with a Yokagawa CSU-X1M 5000 spinning drive system using a Zeiss PlanApochromatic.