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Janeliafluor 646 halotag ligand

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JaneliaFluor 646 HaloTag ligand is a fluorescent dye that can be used to label HaloTag fusion proteins. The ligand binds covalently to the HaloTag protein, allowing for the visualization and detection of the tagged protein of interest.

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Lab products found in correlation

Janelia fluor 549 halotag ligand, by Promega (5 mentions) Dmem f12 medium, by Thermo Fisher Scientific (2 mentions) Axio observer d1 microscope, by Zeiss (2 mentions) Alpha plan apochromat 100 1.46 oil immersion objective, by Zeiss (2 mentions) Evolve 512, by Zeiss (2 mentions) Hydrocortisone h4001, by Merck Group (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) Laminin, by Merck Group (1 mentions) Dmem f12 medium without phenol red, by Thermo Fisher Scientific (1 mentions) Universal detection kit, by American Type Culture Collection (1 mentions) Lipod293, by SignaGen (1 mentions) Attofluor cell chamber, by Thermo Fisher Scientific (1 mentions) Gaspak ez anaerobic pouch, by BD (1 mentions) Deltavision elite microscope, by GE Healthcare (1 mentions) Tri gas incubator, by Panasonic (1 mentions) Ab180520, by Abcam (1 mentions) Ab3612, by Abcam (1 mentions) Gfp trap a, by Proteintech (1 mentions) Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions) Hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)

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HaloTag ligand (16 citations) JaneliaFluor® HaloTag Ligands (2 citations)

17 protocols using janeliafluor 646 halotag ligand

1

Visualizing HaloTagged AP-2 in Eps15 Knockout Cells

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Human-derived SUM159 cells gene-edited to add a HaloTag to both alleles of AP-2 σ2 were a gift from T. Kirchhausen54 (link). Cells were further gene-edited to knock out both alleles of endogenous Eps15 using CRISPR-associated protein 9 (Cas9) to produce the Eps15 knockout cells developed previously by our group12 (link).
Cells were grown in 1:1 DMEM high glucose: Ham’s F-12 (Hyclone, GE Healthcare) supplemented with 5% fetal bovine serum (Hyclone), Penicillin/Streptomycin/l-glutamine (Hyclone), 1 μg ml−1 hydrocortisone (H4001; Sigma-Aldrich), 5 μg ml−1 insulin (I6634; Sigma-Aldrich) and 10 mM HEPES, pH 7.4 and incubated at 37 °C with 5% CO2. Cells were seeded onto acid-washed coverslips at a density of 3 × 104 cells per coverslip for 24 h before transfection with 1μg of plasmid DNA using 3 μl Fugene HD transfection reagent (Promega). HaloTagged AP-2 σ2 was visualized by adding Janelia Fluor 646- HaloTag ligand (Promega). Ligand (100 nM) was added to cells and incubated at 37 °C for 15 min. Cells were washed with fresh medium and imaged immediately.
Yuan F., Gollapudi S., Day K.J., Ashby G., Sangani A., Malady B.T., Wang L., Lafer E.M., Huibregtse J.M, & Stachowiak J.C. (2023). Ubiquitin-driven protein condensation promotes clathrin-mediated endocytosis. bioRxiv.
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2

Culturing and Labeling Neuronal Cell Lines

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Cath.-a-differentiated (CAD) cells were purchased from Sigma-Aldrich and cultured in DMEM/F12 medium (Gibco) supplemented with 8% fetal calf serum, 1% L-Glutamine, and 1% penicillin-streptomycin. Two to four hours prior to imaging, CAD cells were plated on coverslips coated overnight at 4 °C with 10 µg/mL laminin (Sigma-Aldrich). DMEM/F12 medium without phenol red (Gibco) supplemented with 15mM HEPES was used for live-cell imaging. Cell lines were also routinely tested for mycoplasma using the Universal Detection Kit (ATCC). PFN1 KO cells were generated with CRISPR/Cas9 as previously described (28 (link)). CAD cells were transfected with plasmid DNA via electroporation as previously described (28 (link)) or with LipoD293 (SignaGen, “Hard-To-Transfect Mammalian Cell” protocol). Cells expressing HaloTag constructs were incubated overnight with 10–100 nM Janelia Fluor 646 HaloTag ligand (Promega) (61 (link)). EGFP-NM2A knock-in MEFs were generated from mice (62 (link)), and isolated and cultured as previously described (40 (link)).
Vitriol E.A., Quintanilla M.A., Tidei J.J., Troughton L.D., Cody A., Cisterna B.A., Jane M.L., Oakes P.W, & Beach J.R. (2023). Non-muscle myosin 2 filaments are processive in cells. bioRxiv.
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3

Live-Cell Single-Molecule Tracking with Janelia Fluor Dyes

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An Axio Observer D1 Microscope equipped with an Alpha Plan-Apochromat 100 × /1.46 Oil immersion Objective (Zeiss, Germany) and an Evolve 512 × 512 EMCCD camera with pixel size 16.0 μm was used in live-cell SMT experiments. For tracking experiments, an additional 2.5 × magnification was equipped on the emission pathway. A Solid-state LaserStack (3i) was used to excite Janelia Fluor® 549 HaloTag® Ligand (Promega; GA1110) at 552 nm, and Janelia Fluor® 646 HaloTag® Ligand (Promega; GA1120) at 640 nm, respectively. To avoid stray-light reflection and reduce background from cell auto-fluorescence, the HILO illumination model was used. A Brightline® single-band laser filter set (Semrock; excitation filter: FF01–561/14, emission filter: FF01–609/54, and dichroic mirror: Di02-R561–25 3 36) was used for the excitation and emission spectra of JF549 and HaloTag® TMR ligand. A Brightline® single-band laser filter set (Semrock; excitation filter: BLP01–635R-25, emission filter: FF01–640/14–25, and dichroic mirror: Di02-R635–25 3 36) for the excitation and emission spectra of JF646. To filter the excitation wavelength, a TIRF laser microscope cube (3i) was used. The microscope and EMCCD camera were controlled by the computer via SlideBook 6.0 software.
Kent S., Brown K., Yang C.H., Alsaihati N., Tian C., Wang H, & Ren X. (2020). Phase-Separated Transcriptional Condensates Accelerate Target-Search Process Revealed by Live-Cell Single-Molecule Imaging. Cell reports, 33(2), 108248.
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4

Live Imaging of Halo-Tagged Delta-Giardin

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Cells were chilled with ice for 15 minutes to detach from the culture tube and then placed into an Attofluor cell chamber (Molecular Probes) and incubated in a GasPak EZ anaerobic pouch (BD) or a Tri-gas incubator (Panasonic) set to 2.5% O2, 5% CO2 for 90 minutes at 37°C. For live imaging of Halo-tagged Delta-Giardin JaneliaFluor 646 HaloTag ligand (Promega, GA1120) or JaneliaFluor 549 HaloTag ligand (Promega, GA1110) were added to Attofluor cell chambers in growth media at a ratio of 1:1000 during the initial re-attachment step. Cells were then washed four times with HEPES-buffered saline (HBS: 137mM NaCl, 5mM KCl, 0.91mM Na2HpO4-heptahydrate, 5.55mM Glucose, 20mM HEPES, pH7). Live cell imaging was performed on a DeltaVision Elite microscope (GE) equipped with DIC optics, using a 100 × 1.4 NA or 60 × 1.42 NA objective, and a sCMOS 5.4 PCle air-cooled camera (PCO-TECH) and an Oko lab Bold Line stage-top, incubator, set to 37°C, 2.5% O2, and 5% CO2.
Steele-Ogus M.C., Obenaus A.M., Sniadecki N.J, & Paredez A.R. (2022). Disc and Actin Associated Protein 1 influences attachment in the intestinal parasite Giardia lamblia. PLoS Pathogens, 18(3), e1010433.
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5

Antibody Characterization for Diverse Targets

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The following antibodies were used in this study: Polyclonal rabbit anti-GFP (TP401; Torrey Pines; 1:2,000 for western blotting and 1:200 for immunostaining), anti-Rab10 (D36C4) Rabbit mAb (#8127, Cell signaling, 1:1000 for western blotting), anti-Rab11 antibody (ab3612, abcam, 1:1000 for western blotting), Recombinant anti-SNX5 antibody (ab180520, abcam, 1:1000 for western blotting). Purified Mouse anti-SNX1 (611482, BD biosciences, 1:100 for IF), anti-SNX6 mouse monoclonal antibody (D-5) (sc-365965, Santa Cruz, 1:1000 for western blotting), For pulldowns, Trap beads (nanobodies) were used. GFP-Trap_A (chromotek, gta-20) was used for GFP pulldowns. HRP-conjugated goat anti-mouse IgG (H  +  L) secondary antibody (ThermoFisher Scientific; 31430; 1:10,000) and polyclonal HRP-conjugated goat anti-rabbit IgG (ThermoFisher Scientific; 31460; 1:10,000) were used (incubated for 1 h at room temperature) to detect bound antibodies with Blotting detection kit WesternBrightTM ECL (advansta; K-12045-D50). Alexa Fluor 488–goat anti-rabbit IgG (H  +  L) (Invitrogen; A-11034) and Alexa Fluor 594–goat anti-mouse IgG (H  +  L) cross-adsorbed secondary antibodies (Invitrogen; R37121) were used for immunofluorescence. Janelia Fluor® 646 HaloTag® Ligand (Promega; Catalog number GA1120) was used for cargo transfer experiments.
Solinger J.A., Rashid H.O, & Spang A. (2022). FERARI and cargo adaptors coordinate cargo flow through sorting endosomes. Nature Communications, 13, 4620.
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6

Live-Cell Single-Molecule Tracking with Janelia Fluor Dyes

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An Axio Observer D1 Microscope equipped with an Alpha Plan-Apochromat 100 × /1.46 Oil immersion Objective (Zeiss, Germany) and an Evolve 512 × 512 EMCCD camera with pixel size 16.0 μm was used in live-cell SMT experiments. For tracking experiments, an additional 2.5 × magnification was equipped on the emission pathway. A Solid-state LaserStack (3i) was used to excite Janelia Fluor® 549 HaloTag® Ligand (Promega; GA1110) at 552 nm, and Janelia Fluor® 646 HaloTag® Ligand (Promega; GA1120) at 640 nm, respectively. To avoid stray-light reflection and reduce background from cell auto-fluorescence, the HILO illumination model was used. A Brightline® single-band laser filter set (Semrock; excitation filter: FF01–561/14, emission filter: FF01–609/54, and dichroic mirror: Di02-R561–25 3 36) was used for the excitation and emission spectra of JF549 and HaloTag® TMR ligand. A Brightline® single-band laser filter set (Semrock; excitation filter: BLP01–635R-25, emission filter: FF01–640/14–25, and dichroic mirror: Di02-R635–25 3 36) for the excitation and emission spectra of JF646. To filter the excitation wavelength, a TIRF laser microscope cube (3i) was used. The microscope and EMCCD camera were controlled by the computer via SlideBook 6.0 software.
Kent S., Brown K., Yang C.H., Alsaihati N., Tian C., Wang H, & Ren X. (2020). Phase-Separated Transcriptional Condensates Accelerate Target-Search Process Revealed by Live-Cell Single-Molecule Imaging. Cell reports, 33(2), 108248.
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7

Protein Analysis and Transfection Assays

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The DC Protein Assay (500-0119) and Clarity Western ECL Substrate (170-5061) were from BioRad. The Effectene Transfection Reagent (301427) was from Qiagen. The Lipofectamine RNAiMAX Reagent (100014472) was from Thermo Fisher Scientific. ProLong Diamond antifade mountant without (P36970) or with (P36971) DAPI (4′,6-diamidino-2-phenylindole) was from Thermo Fisher Scientific. BafA1 (11038) and MRT68921 (1990520000040) were from Cayman Chem. PIK-III was from Selleckchem (S7683). Bort (B-1408) was from LC Laboratories. Janelia Fluor 646 HaloTag Ligand (GA1120) was from Promega.
Plasmids used and sources are given in Table 1. Antibodies used along with hosts and manufacturers appear in Table 2.
Kennedy A., Ren H.Y., Madden V.J, & Cyr D.M. (2022). Lysosome docking to WIPI1 rings and ER-connected phagophores occurs during DNAJB12- and GABARAP-dependent selective autophagy of misfolded P23H-rhodopsin. Molecular Biology of the Cell, 33(9), ar84.
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8

Immunostaining of Cells Expressing HaloTag Fusion Proteins

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COS-7 or HeLa cells were grown on #1.5 coverslips (Thermo Fisher Scientific; 12541A) in six-well plates. After transfection and treatment, conditions with HaloTag fusion protein expression were also incubated with 100 nM Janelia Fluor 646 HaloTag Ligand (Promega; GA11220) for 30 min before fixation. Cells were fixed at –20°C for 5 min with precooled methanol. Cells were rehydrated by being washed 3× in PBS and blocked with 3% bovine serum albumin (BSA) in PBS for 30 min before immunostaining with various antibodies in 3% BSA in PBS for 1 h. Goat anti-mouse or goat anti-rabbit secondary antibodies with different fluorophores were used to detect proteins of interest in 3% BSA in PBS for 20 min. Coverslips were washed 8× with PBS, mounted onto slides using ProLong Diamond antifade mountant without (P36970) or with (P36971) DAPI (Invitrogen), and then sealed with nail polish. Slides were allowed to dry in the dark at room temperature for at least 12 h before imaging.
Kennedy A., Ren H.Y., Madden V.J, & Cyr D.M. (2022). Lysosome docking to WIPI1 rings and ER-connected phagophores occurs during DNAJB12- and GABARAP-dependent selective autophagy of misfolded P23H-rhodopsin. Molecular Biology of the Cell, 33(9), ar84.
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9

Immunocytochemistry Assay for NCI-H441 Cells

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Immunocytochemistry assays were performed using fixed cells on round cover glass, #1.5 thickness, 10 mm (Thomas Scientific, 1217N78). NCI-H441 cells were grown on the coverslips and fixed for 10 min with 3.7% paraformaldehyde and quenched with 10 mM ammonium chloride. Cells were then permeabilized with 0.1% Triton X-100 in PBS for 10 min. The coverslips were then washed with PBS and blocked in 1XPBS, 2.5% goat serum (Sigma), 0.2% Tween 20 for 10 min followed by 10 min blocking in PBS, 0.4% fish skin gelatin (Sigma), and 0.2% Tween 20. Cells were incubated with primary antibodies for 1 h at room temperature. The coverslips were then washed with PBS, 0.2% Tween 20, and incubated with Alexa Fluor 594 or 647 secondary antibodies for 45 min, washed as described above, and mounted on glass slides in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific-P36965). IF imaging involving fixed cell imaging was treated with Janelia Fluor 646 HaloTag Ligand (Promega catalogue # GA1120) according to manufacturer’s guidelines.
Awadia S., Sitto M., Ram S., Ji W., Liu Y., Damani R., Ray D., Lawrence T.S., Galban C.J., Cappell S.D, & Rehemtulla A. (2023). The adapter protein FADD provides an alternate pathway for entry into the cell cycle by regulating APC/C-Cdh1 E3 ubiquitin ligase activity. The Journal of Biological Chemistry, 299(6), 104786.
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10

Labeling Halo-tagged RPA2 for Microscopy

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Cells with endogenously tagged Halo::RPA2 were labelled with 200 nm of Janelia Fluor 646 HaloTag ligand (JF646; Promega; GA1120) by replacing one-fifth of the culture media with 1 µM JF646. Cells were then incubated for 30 min at 37 °C and harvested for microscopy after fixation in media. For combined antibody staining with Halo::RPA2 imaging, samples were permeabilised with 0.1% NP-40 for 2 min and processed as described above.
Budzak J., Jones R., Tschudi C., Kolev N.G, & Rudenko G. (2022). An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes. Nature Communications, 13, 101.
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