Cells expressing SNAP-β2AR or SNAP-GPI were labeled with 20 nM Lumi4-Tb and 80 nM SNAP-red (Cisbio, Codolet, France) for 2 hours at 37°C in complete growth medium. Cells were washed three times in PBS, harvested by trituration, and transferred to white opaque 96-well plates. Time-resolved FRET was measured using a Biotek (Winooski, VT) Synergy 2 plate reader using a xenon flash lamp. Filters used for excitation, donor emission and acceptor emission were 330/80 nm, 620/10 nm and 665/7 nm, respectively, and emission was integrated for 10 ms starting 150 μs after each excitation flash. Background and donor bleedthrough were negligible at this 665 nm, and direct acceptor excitation was undetectable after 150 μs, therefore Em 665 corresponds essentially to sensitized emission.
Snap red
Manufactured by PerkinElmer
Sourced in France
SNAP-red is a fluorescent labeling reagent designed for the selective and covalent labeling of SNAP-tag fusion proteins. It allows for the detection and visualization of SNAP-tag fusion proteins in various applications, such as live-cell imaging and in-gel fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Snap lumi4 tb, by PerkinElmer (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Ly341495, by Bio-Techne (2 mentions)
Ly379268, by Bio-Techne (2 mentions)
Snap green, by PerkinElmer (2 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Lumi4 tb, by PerkinElmer (1 mentions)
Bodipy fl gtpγs, by Thermo Fisher Scientific (1 mentions)
Glutamate, by Merck Group (1 mentions)
Quisqualate, by Bio-Techne (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Ex cell 420 medium, by Merck Group (1 mentions)
Snap alexa fluor 488, by New England Biolabs (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dcg 4, by Bio-Techne (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Ly487379, by Bio-Techne (1 mentions)
Quikchange mutagenesis protocol, by Agilent Technologies (1 mentions)
Facscalibur, by BD (1 mentions)
7 protocols using snap red
1
Time-Resolved FRET Analysis of SNAP-β2AR
2
Radiolabeled MPEP Binding Assay
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Tritiated 2-methyl-6-(phenylethynyl)pyridine hydrochloride [3H] MPEP was purchased from the American Radiolabeled Chemicals incorporation (ARC), glutamate from Sigma Aldrich, quisqualate and MPEP from Tocris. Sf9 cells adapted in SF-900 II SFM and EX-CELL-420 medium were ordered from Sigma Aldrich, lipofectamine 2000 and DMEM medium from Life Technologies. Detergents were ordered from Anatrace. SNAP-Lumi4-Tb and SNAP Red were obtained from Cisbio Bioassays. Bodipy-FL GTPγS was purchased from ThermoFisher. (R)-5-((3-fluorophenyl) ethynyl)-N-(3-hydroxy-3-methylbutan-2-yl) picolinamide (VU0424465) was synthetized as detailed in the supplementary materials.
3
Visualizing ST-δ and ST-CXCR7 Receptor Internalization
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Cells expressing ST-δ receptor and ST–CXCR7 expressing cells were seeded in 8-well slides for 24 h. Cell surface ST-δ receptor molecules were labeled during for 30 min with the cell-impermeable O6-benzylguanine derivative cell impermeable SNAP-Alexa Fluor 488 (New England Biolabs, Ipswich, MA, USA) at room temperature. Cells were washed three times with Krebs/Tris buffer and incubated for 30 min with either Tag-lite medium either alone or containing SNC162 (10 μM) or naloxone (10 μM). ST–CXCR7 cells were labeled for 1 h with SNAP-red (Cisbio Bioassays) at 4°C and then warmed to 37°C for 1 h to visualize constitutive internalization. Fluorescence was detected on a Zeiss fluorescent microscope with a xenon flash lamp using a GFP or Cy5 filter cube and a Zeiss Plan Apo 63× (1.40 oil) objective.
4
GPCR Expression and Characterization
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HEK293 cells (ATCC, CRL-1573, lot: 3449904) were cultivated in DMEM (Thermo Fischer Scientific, Courtaboeuf, France) complemented with 10% (v/v) fetal bovine serum. Absence of mycoplasma was routinely checked using the MycoAlert Mycoplasma detection kit (LT07-318, Lonza, Amboise, France), according to the manufacturer protocol. All drugs (DCG-IV, LY341495, LY379268, and LY487379) were from Tocris Bioscience (Bristol, UK). LSP4-2022 was provided by Dr. F. Acher (Paris, France). All HTRF reagents, labeled monoclonal antibodies anti-c-Myc-d2 (61MYCDAA, Cisbio Bioassays, Codolet, France), and anti-6His-d2 (61HISDLA, Cisbio Bioassays), labeled ligands (SNAP-Lumi4-Tb, SNAP-Red, CLIP-Red, and BG-Alexa Fluor 488) were a kind gift from Cisbio Bioassays. The pRK5 plasmids encoding wild-type rat mGluR subunits, with a HA-tag and with SNAP or CLIP inserted just after the signal peptide, were previously described46 (link). pEGFP-C2 plasmid encoding EGFP was from Clontech (Mountain View, CA, USA). Point mutations were introduced in the SNAP-tag mGlu2 or mGlu3 plasmids according to the QuikChange mutagenesis protocol (Agilent Technologies, Santa Clara, CA, USA).
5
Flow Cytometry Analysis of SNAP-Tagged Receptors
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Cells expressing SNAP-tagged receptors were labeled with 80 nM SNAP-red (Cisbio, Codolet, France) for 2 hours at 37° C in complete growth medium. Cells were washed three times in PBS, harvested by trituration, and fixed with 4% paraformaldehyde in PBS. Flow cytometry was performed using a FACSCalibur (BD Biosciences, San Jose, CA) and 488 nm and 633 nm excitation lasers. Venus was detected in FL1 (530/30 nm) and SNAP-red was detected in FL4 (661/16 nm). Gates for background were determined using untransfected cells stained with SNAP-red. Identical excitation and detection amplifier settings were used across all experiments, and data were analyzed with FlowJo (Tree Star, Ashland, OR).
6
Multimodal Binding Assay Protocol
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SNAP-Lumi4-Tb, SNAP-Green, SNAP-Red and BG-Alexa Fluor 488 (BG-Alexa488) were from Cisbio Bioassays (Codolet, France). BINA, LY341495, and LY379268, were purchased from Tocris Bioscience (Bristol, UK). Laminin, dichloro(1,10-phenanthroline)copper(II) (CuP) and salts for preparing buffers were from Sigma-Aldrich (St. Louis, MO, USA).
7
GABA receptor binding assay
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Materials and Reagents GABA, CGP54626, and SKF97541 were purchased from Tocris Bioscience. Baclofen, 3-APPA, CGP35348, CGP7930, GS39783, and rac-BHFF were purchased from Abcam. Lipofectamine 2000 and Fluo-4 AM were from Thermo Fischer Scientific. SNAP-Lumi4-Tb, SNAP-Green, SNAP-Red, CoA-Lumi4-Tb, and Tag-Lite buffer can be purchased from Cisbio Bioassays. CoA-Alexa 488, CoA-647, and Sfp synthase were from New England Biolabs. Benzyl guanine (BG) derivative fluorophores BG-Green-LC and BG-Atto465, and CGP54626-Red were a gift from Cisbio Bioassays.
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