click labeling h tet cy5  (Jena Bioscience)

Journal: Protein Science : A Publication of the Protein Society

Article Title: Optimized genetic code expansion technology for time‐dependent induction of adhesion GPCR ‐ligand engagement

doi: 10.1002/pro.4614

Figure Lengend Snippet: Bioorthogoncal click‐labeling via GCE allows for co‐localization of ADGRE5 and its ligand CD55. (a) Protein layouts in the assays. (b) Co‐incubation of HEK293T cells expressing either E5‐mCitr CTF or CD55‐TAG Ex ‐TM labeled with Tet‐Cy5. Cell–cell contacts show co‐localization of the receptor (yellow; chevrons) and the ligand proteins (cyan [mTurq fluorescence] and magenta [Tet‐Cy5 label]; arrowheads). Note that E5‐mCitr CTF is enriched at cell membrane areas that are in contact with ligand‐presenting cells. Scale bar = 10 μm.

Article Snippet: An automated time series was performed in order to get images at periodic time points over 24 h. For live‐cell imaging via click labeling H‐Tet‐Cy5 (Jena Bioscience #CLK‐015‐05) was used.

Techniques: Labeling, Incubation, Expressing, Fluorescence, Membrane

Journal: Protein Science : A Publication of the Protein Society

Article Title: Optimized genetic code expansion technology for time‐dependent induction of adhesion GPCR ‐ligand engagement

doi: 10.1002/pro.4614

Figure Lengend Snippet: GCE allows for time‐control of ADGRE5‐CD55 engagement in vitro. (a) Surface ELISA of CD55‐TAG Ex ‐TM‐mTurq In at indicated time intervals after addition of TCO*. (b) Comparison of surface CD55‐TAG Ex ‐TM‐mTurq In kinetics co‐expressed with standard (system VI, gray) or GCEXpress (system X, blue) GCE plasmids. Fits calculated with Graphpad Prism v9. (c) Confocal image series showing co‐incubated E5‐mCitr CTF ‐expressing HEK293T cell in contact with a CD55‐TAG Ex ‐TM‐mTurq‐expressing cell (asterisk). Note the enrichment of E5‐mCitr CTF at the membrane area that is interfacing with the CD55‐TAG Ex ‐TM‐mTurq In cell, but not at cell–cell contacts with ligand‐less membranes. Scale bar = 10 μm. (d) Close‐up view of cell–cell contact shown in (C) at 24 h after TCO* feeding. Cells were stained with Tet‐Cy5 (magenta) to confirm CD55‐TAG Ex ‐TM‐mTurq (cyan) residence in apposition to E5‐mCitr CTF membranes. Scale bar = 10 μm.

Article Snippet: An automated time series was performed in order to get images at periodic time points over 24 h. For live‐cell imaging via click labeling H‐Tet‐Cy5 (Jena Bioscience #CLK‐015‐05) was used.

Techniques: Control, In Vitro, Enzyme-linked Immunosorbent Assay, Comparison, Incubation, Expressing, Membrane, Staining

Journal: Nature Methods

Article Title: Photoswitching fingerprint analysis bypasses the 10-nm resolution barrier

doi: 10.1038/s41592-022-01548-6

Figure Lengend Snippet: a , Scheme of DNA origami labeled with four Cy5 at interfluorophore distances of 18 nm, 9 nm, 6 nm and 3 nm. b , c , Selected dSTORM ( b ) and DNA-PAINT ( c ) images of DNA origami. Samples were measured 3–5 times independently. Scale bars, 40 nm. d , Analysis of fluorescence trajectories recorded from individual DNA origami imaged using 640 nm excitation at an intensity of 5 kW cm −2 . e , f , Relative occurrence of fluorescence intensity ms −1 in the on-state (Intensity), lifetime of the on-state (On-time), lifetime of the off-state (Off-time) and number of on-states (On-events) detected for DNA origami with different interfluorophore distances in dSTORM ( e ) calculated from n = 3–5 and DNA-PAINT ( f ) calculated from n = 2–3 individual experiments. Color code; singly labeled reference (gray), 18 nm (dark blue), 9 nm (light blue), 6 nm (red) and 3 nm (orange). g , Number of on-events (cumulative localizations, cum. locs.) detected per frame as a function of time during 10 min dSTORM videos (Supplementary Videos – ) of DNA origami with different interfluorophore distance ( n = 3–5). h , Histogram of the times after which 80% of all localizations were detected per individual DNA origami ( n = 3–5).

Article Snippet: Transfected HEK293T expressing the TCO*-A modified GluK2, or GABA-A α2 or GABA-A γ2 receptor subunits were labeled with 3 μM tetrazine coupled fluorophores H-Tet-Cy5 (Jena Bioscience, no. CLK-015-05) in cell growth medium for 60 min on ice.

Techniques: Labeling, Fluorescence

Journal: Nature Methods

Article Title: Photoswitching fingerprint analysis bypasses the 10-nm resolution barrier

doi: 10.1038/s41592-022-01548-6

Figure Lengend Snippet: a – e , Fluorescence trajectories recorded for a singly labeled reference ( a ), 18 nm ( b ), 9 nm ( c ), 6 nm ( d ) and 3 nm ( e ) DNA origamis in dSTORM photoswitching buffer. Color code, singly labeled reference (gray), 18 nm (dark blue), 9 nm (light blue), 6 nm (red) and 3 nm (orange). Zoomed-in trajectories of the first seconds show fast blinking observed for the 6- and 3-nm DNA origamis. Time bins, 1 ms. f , Fluorescence trajectory recorded for a 3-nm DNA origami in trolox buffer and zoomed-in fluorescence signal of the first 2 s. Time bin, 1 ms. g , Average fluorescence decays from n = 7–10 individual fluorescence trajectories of singly labeled reference (gray) and 3-nm DNA origamis measured in trolox (black) and photoswitching buffer (orange), respectively, revealing different energy transfer pathways between the Cy5 fluorophores. h , Average intensity autocorrelation functions (G( τ )) calculated from n = 7–10 individual fluorescence trajectories of singly labeled reference and 3-nm DNA origamis measured in trolox and photoswitching buffer, respectively, normalized to 1 ms. i , Histogram of average FLIMs measured from n = 7–15 fluorescence trajectories of individual DNA origami with different interfluorophore distances of 18, 9, 6 and 3 nm in photoswitching buffer. j , Fluorescence trajectory of a 3 nm DNA origami in photoswitching and corresponding fluorescence decays with average fluorescence lifetimes ( τ AV ) of 0.66, 1.25 and 1.77 ns recorded during the gray marked areas. k , Typical FLIM images of the 18-, 9-, 6- and 3-nm DNA origami measured in trolox buffer emphasize the increased blinking and shorter fluorescence lifetime of Cy5 fluorophores in the sub-10-nm range (moving from top left to the bottom right). The samples were measured 5–10 times and excited at 640 nm with 2.5 kW cm −2 at an integration time of 5 µs pixel −1 . Scale bar, 1 µm.

Article Snippet: Transfected HEK293T expressing the TCO*-A modified GluK2, or GABA-A α2 or GABA-A γ2 receptor subunits were labeled with 3 μM tetrazine coupled fluorophores H-Tet-Cy5 (Jena Bioscience, no. CLK-015-05) in cell growth medium for 60 min on ice.

Techniques: Fluorescence, Labeling

Journal: Nature Methods

Article Title: Photoswitching fingerprint analysis bypasses the 10-nm resolution barrier

doi: 10.1038/s41592-022-01548-6

Figure Lengend Snippet: a – c , Molecular structures of the pentameric GABA-A (PDB 6HUG ) and tetrameric GluK2 receptor (PDB 5KUF ) with incorporation sites of ncAAs shown as black circles (blue, γ2 subunit GABA-A ( a ); red, dimeric α2 GABA-A ( b ); orange, homotetrameric GluK2 ( c )) and corresponding dSTORM images of HEK293T membrane sections showing fluorescence signals of individual receptors (5 nm pixel −1 ). The ncAAs were labeled by click chemistry with Met-Tet-Cy5. In the GABA-A S181TAG mutant the distance between the two fluorophores in the α2 subunits is roughly 5 nm. In the GluK2 S398TAG mutant the distance between the four Cy5 molecules is roughly 7 nm (refs. , ). The samples were measured 3–5 times independently. Scale bars, 500 nm. d , Relative occurrence of lifetimes of the off-state (Off-time), and number of on-states (On-events) detected from individual receptors in dSTORM experiments ( n = 3–5). e , Number of on-events (localizations) detected per frame as a function of time during 10 min dSTORM experiments of membrane receptors ( n = 3–5). f , FLIM images of HEK293T cells expressing monomeric γ2 subunit of GABA-A (left, blue), dimeric α2 subunit of GABA-A (middle, red), and homotetrameric GluK2 receptors (right, orange) click-labeled with Met-Tet-Cy5 measured by confocal TCSPC imaging in photoswitching buffer at an irradiation intensity of 2.5 kW cm −2 . To minimize photobleaching of fluorophores FLIM images were recorded at 5 µs of integration time per pixel. No intensity threshold was applied. Scale bars, 2 µm. g , Average fluorescence decays from n = 8–13 FLIM images of HEK293T cells expressing receptors labeled with one, two and four Cy5 fluorophores.

Article Snippet: Transfected HEK293T expressing the TCO*-A modified GluK2, or GABA-A α2 or GABA-A γ2 receptor subunits were labeled with 3 μM tetrazine coupled fluorophores H-Tet-Cy5 (Jena Bioscience, no. CLK-015-05) in cell growth medium for 60 min on ice.

Techniques: Fluorescence, Labeling, Mutagenesis, Expressing, Imaging, Irradiation

Journal: Nature Methods

Article Title: Photoswitching fingerprint analysis bypasses the 10-nm resolution barrier

doi: 10.1038/s41592-022-01548-6

Figure Lengend Snippet: a , Different mutants 1–4 were generated within one monomeric subunit of GluK2. Calculation of the distances was performed with PyMOL (Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC) on basis of the crystal structure (PDB-ID: 5KUF)41,42. b , To check the efficiency of ncAA incorporation of the different mutants, click labeling was performed with H-Tet-Cy5. Control experiments without the addition of ncAA resulted in inefficient amber suppression efficiency which leads to premature translation termination and no click labeling. Samples were measured 3–5 times independently. Scale bars, 10 µm.

Article Snippet: Transfected HEK293T expressing the TCO*-A modified GluK2, or GABA-A α2 or GABA-A γ2 receptor subunits were labeled with 3 μM tetrazine coupled fluorophores H-Tet-Cy5 (Jena Bioscience, no. CLK-015-05) in cell growth medium for 60 min on ice.

Techniques: Generated, Labeling

Journal: Nature Communications

Article Title: Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids unveils masked epitopes in live neurons

doi: 10.1038/s41467-021-27025-w

Figure Lengend Snippet: a Depiction of the workflow used for expression of ncAA-tagged TARPs in CA1 pyramidal cells in OHSC using single-cell electroporation (SCE), and live staining with tetrazine-dyes. b Example confocal image of fixed CA1 neurons co-expressing eGFP and γ2 S44* in OHSC. Images are projections of a z-stack taken by 1 μm increments, eGFP signal is color-coded with respect to sample depth. c , e Representative confocal images of CA1 neurons co-expressing eGFP, and c γ2 S44*- or h γ8 S72* live stained with 1 μM H-Tet-Cy5. d , f Magnified views of segments of the basal and apical dendrites from d γ2 S44*- and f γ8 S72*-overexpressing CA1 neurons highlighted in the corresponding overview images (yellow boxes). g , i Close up of representative spines from g γ2 S44*- and i γ8 S72*-overexpressing CA1 neurons highlighted (dashed squares) in the overview images ( d ) and ( f ), respectively. h , j Line scan measurements of Cy5 signal across spines in g and i respectively. k , l Confocal images of segments of basal dendrites from CA1 neurons co-expressing either k γ2 S44* or l γ8 S72, and the PSD-95 marker, XPH20::eGFP. Bottom insets: line scans of the GFP and Cy5 signal for the 3 μm segments indicated in the above images. Scale bar: ( b , c , h ) 100 μm and ( d , g , i , l ) 5 μm. ( b – e , h – j ) example images are representative of three or four independent preparations, and ( g , l ) from two independent preparations.

Article Snippet: Pyrimidyl-Tetrazine-Alexa Fluor 647 (Pyr-Tet-AF647; #CLK-102), Pyr-Tet-ATTO-643 (Pyr-Tet-ATTO643; #CLK-101), H-Tet-Cy3 (#CLK-014-05), and H-Tet-Cy5 (#CLK-015-05) were purchased from Jena Bioscience (Jena, Germany).

Techniques: Expressing, Electroporation, Staining, Marker