HEK293T cells grown on 10cm plates were checked for approximately 50% confluency. The cells were then transfected with 20 μg DNA per 10 cm plate following JetPrime protocol. DMEM media was changed after four hours of transfection and was left for overnight expression. Next day, cells were collected from two transfected 10 cm plates and were washed with 3mL of extracellular buffer (ECB). The sample was then wrapped in foil and was labeled with 400 nM of Alexa 555 maleimide (ThermoFisher), a donor fluorophore and 400 nM of Alexa 647 maleimide (ThermoFisher), an acceptor fluorophore, in 3mL ECB at room temperature for 1hr. The labelled cells were washed with 3mL ECB and were resuspended in 2mL of solubilization buffer by nutating at 4°C for one hour. Solubilization buffer consists of phosphate-buffered saline, 1% lauryl maltose neopentyl glycol (Anatrace), 2 mM cholesteryl hydrogen succinate (MP Biomedicals), and 1/4 protease inhibitor tablet (Pierce). The nutated sample was then transferred to an ultracentrifuge tube and was spun for one hour at 44000 rpm at 4°C using a TLA 100.3 rotor for filtering unsolubilized debris. Supernatant thus collected were used as smFRET samples and were kept on ice until they were used.
Alexa 647 maleimide
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 647 maleimide is a fluorescent dye used for labeling biomolecules. It contains a maleimide reactive group that can form a covalent bond with sulfhydryl groups, allowing for site-specific labeling of proteins and other biomolecules.
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Lab products found in correlation
Alexa 555 maleimide, by Thermo Fisher Scientific (8 mentions)
Cholesteryl hydrogen succinate, by MP Biomedicals (5 mentions)
Lauryl maltose neopentyl glycol, by Anatrace (5 mentions)
Dl ap5, by Abcam (4 mentions)
Acrylodan, by Thermo Fisher Scientific (2 mentions)
Alexa 488 maleimide, by Thermo Fisher Scientific (2 mentions)
Tissue tek oct, by Thermo Fisher Scientific (1 mentions)
1 2 dioleoyl sn glycero 3 phospho l serine dops, by Avanti Polar Lipids (1 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine popc, by Avanti Polar Lipids (1 mentions)
Alexa 555, by Thermo Fisher Scientific (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Alexa555, by GE Healthcare (1 mentions)
Cholesterol, by Avanti Polar Lipids (1 mentions)
L α phosphatidylinositol 4 5 bisphosphate, by Avanti Polar Lipids (1 mentions)
Nickel nitrilotriacetic acid agarose, by Qiagen (1 mentions)
Pd 10, by GE Healthcare (1 mentions)
Pbs buffer, by GE Healthcare (1 mentions)
Human egf, by Thermo Fisher Scientific (1 mentions)
18 protocols using alexa 647 maleimide
1
Fluorescent Labeling and Solubilization of HEK293T Cells
2
Transient Transfection and Labeling of HEK293T Cells for smFRET
3
Fluorescent Labeling of HEK 293T Cells
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HEK 293T cells were transiently transfected using the JetPrime protocol at 10 µg per 10-cm plate. One day after transfection, the cells from two 10-cm dishes were harvested; washed with extracellular buffer (ECB) containing 135 mM NaCl or CsCl, 3 mM KCl, 2 mM CaCl2, 20 mM glucose, and 20 mM HEPES; and adjusted to pH 7.4 with NaOH or CsOH. Post-wash, the cells were labeled for 1h at room temperature with 600 nM of donor fluorophore Alexa 555 maleimide (ThermoFisher, Waltham, MA, USA) and 2.4 µM of acceptor fluorophore Alexa 647 maleimide (ThermoFisher) in 3 mL of ECB. Post-labeling, the cells were washed and solubilized for 1 h at 4 °C. Solubilization buffer contained 1% lauryl maltose neopentyl glycol (Anatrace, Maumee, OH, USA), 2 mM cholesteryl hydrogen succinate (MP Biomedicals, Irvine, CA, USA), and ¼ protease inhibitor tablet (Pierce™) in phosphate-buffer saline containing either NaCl and Na2HPO4 for experiments in the presence of Na+ or CsCl and K2HPO4 for experiments in the absence of Cs+. Solubilized cells were filtered from unsolubilized debris by ultracentrifugation at 100,000× g for 1 h at 4 °C using a TLA 100.3 rotor. The supernatant was collected and kept on ice until used for smFRET samples.
4
Transient Transfection and Labeling of HEK293T Cells for smFRET
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HEK293T cells were transiently transfected with GluN1*F554C and GluN2A* DNA at a mass ratio of 2.5:7.5 μg per 10 cm dish. An equivalent mass of GluN1*F554C-TS was used in place of GluN1*F554C for experiments involving it. 300 μM DL-AP5 (abcam) and 30 μM DCKA (abcam) were present during transfection to limit excitotoxicity. One day post-transfection, cells from two 10-cm dishes were harvested and labeled for 1 hour at room temperature with 150 nM of donor fluorophore Alexa 555 maleimide (ThermoFisher) and 600 nM of acceptor fluorophore Alexa 647 maleimide (ThermoFisher) in 3 mL extracellular buffer. After washing, labeled cells were then solubilized for 1 hour at 4°C in buffer containing phosphate-buffered saline, 1% lauryl maltose neopentyl glycol (Anatrace), 2 mM cholesteryl hydrogen succinate (MP Biomedicals), and protease inhibitor (Pierce). Unsolubilized debris were then spun down for 1 hour at 100,000 × g at 4°C, and the supernatant used as the smFRET sample.
5
Redox Staining of Brain Tissue
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For redox staining (protocol modified from Horowitz et al.15 (link)), the excised brains were placed in a solution containing 4% paraformaldehyde, 1 mM N-ethyl maleimide (Sigma-Aldrich E3876), 2 µM Alexa647-maleimide (Thermo Fischer A20347) and 0.05% Triton X-100 (Sigma-Aldrich X-100) in PBS for incubation overnight (ca. 16 h) at 8 °C. Then, the brains were rinsed in PBS (4 × 30 min), incubated in 5 mM TCEP (Sigma-Aldrich 75259) in PBS for 6 h, rinsed in PBS (5 × 15 min), and placed in a solution containing 1 mM N-ethyl maleimide and 2 µM Alexa555-maleimide (Thermo Fischer A20346) in PBS for incubation overnight (ca. 16 h) at 8 °C. Then, the brains were rinsed in PBS, cryoprotected in PBS with 25% (w/v) sucrose, and frozen on dry ice in TissueTek OCT (Fisher Scientific: epredia Neg-50). The brains were cryosectioned in the sagittal plane (30 µm section thickness). Sections were mounted on Superfrost Gold slides and coverslipped with ProLong Gold antifade reagent with DAPI (Thermo Fischer P36931) as the mounting medium. In the presented figures, fluorescence microscopy of the sections shows the extent of oxidized thiols (magenta), indicating tissue damage against a background of reduced thiols (cyan).
6
Preparation of Mutant FtsY Proteins
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Plasmids for mutant FtsY were constructed using the QuikChange mutagenesis protocol (Stratagene). The expression construct for His6-FtsY-dN1 was a kind gift from the Schaffitzel Lab. mRNAs for in vitro translations were synthesized by in vitro transcription using T7 (for RNC preparation) or SP6 (for co-translational translocation assay) polymerases following the Megascript protocol (Ambion). Wildtype and mutant Ffh and FtsY and 4.5S RNA were expressed and purified as described in previous studies (Peluso et al., 2001 (link); Lam et al., 2010 (link)). RNCs bearing signal sequences of FtsQ or Luc were prepared as described previously (Zhang et al., 2010 (link)). FtsY-C345 and Ffh-C153 were labeled with Alexa647-maleimide (Thermo Fischer, Waltham, MA) and FtsY-C356 was labeled with acrylodan (Invitrogen) as described (Shen et al., 2012 (link)) with the minor modifications. Details see the SI Methods. All proteins were exchanged into SRP buffer (50 mM HEPES-KOH, pH 7.5, 150 mM KOAc, 10 mM Mg(OAc)2, 2 mM dithiothreitol (DTT) and 0.01% octaethyleneglycol dodecylether (Nikkol)) prior to use.
7
Nmdar Labeling and Functional Characterization
8
Synaptotagmin-1 C2AB Domain Labeling
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Adenosine phosphates (ATP, ADP) and Inositol hexaphosphate (IP6) were purchased from Sigma-Aldrich (St Louis, MO). Inositol 1,4,5-trisphosphate (IP3), phosphatidylinositol 4,5-bisphosphate diC4 (PIP2-diC4) were purchased from Echelon biosciences (Salt Lake City, UT). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) were purchased from Avanti Polar lipids (Alabaster, AL). Thiol reactive fluorescent probes Alexa488-maleimide and Alexa647-maleimide were purchased from Thermo Scientific, Waltham, MA. The DNA constructs used in this study - the wild-type C2AB domain (Syt1C2AB, residues 143–421) of rat Synatotagmin-1, C2B polylysine mutant (Syt1C2ABK326A, K327A), C2B Ca2+ binding mutant (Syt13A, D309A, D363A, D365A) were generated and sequenced in our earlier works (Wang et al., 2014 (link); Zanetti et al., 2016 (link)). For site-specific labeling with fluorophores, cysteine was introduced in Syt1C2AB at residue 269, while naturally existing cysteine at residue 277 was removed (C277S) using the Quickchange mutagenesis kit (Stratagene, Santa Clara, CA)
9
Nmdar Labeling and Functional Characterization
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HEK293T cells were plated into 30 mm dishes and transfected (jetPrime, PolyPlus) with GluN1*F554C and GluN2A* constructs at a mass ratio of 1.5:4.5 μg per 20 ml of media. 300 μM DL-AP5 (abcam) and 30 μM DCKA (abcam) were present in the media and recordings were performed 24 to 48 hours post-transfection. Prior to recording, cells were incubated in 150 nM Alexa 555 maleimide (ThermoFisher) and 600 nM Alexa 647 maleimide (ThermoFisher) for at least 1 hour to mimic smFRET labelling conditions. Outside-out patches were excised and piezo-driven solution exchange was performed as outlined elsewhere51 (link). The external solution was (in mM) 150 NaCl, 20 HEPES, 10 Tricine, 1 CaCl2, and 0.1 glycine, pH 7.4 (NaOH). The pipette solution was 135 CsF, 33 CsOH, 11 EGTA, 10 HEPES, 2 MgCl2 and 1 CaCl2, pH 7.4. Lifted whole cell recordings were performed as described elsewhere52 (link) using the same solutions as above with the addition of 2.5 mM KCl to the external solution.
10
Stochastic Labeling of OpuA Protein
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Stochastic labeling was performed with the dyes Alexa555 and Alexa647 maleimide (Thermo Fisher Scientific). OpuA (5 to 10 nmol) was first treated with 10 mM DTT for 10 min to fully reduce oxidized cysteines. After dilution of the protein sample to a DTT concentration of 1 mM, the reduced protein was immobilized on 200 μl of Ni2+-Sepharose resin and washed with 2 ml of buffer A [20 mM K-Hepes (pH 7.0) and 300 mM KCl] to remove the DTT. The resin was incubated in 1 ml of buffer A supplemented with 50 nmol of Alexa555 and 50 nmol of Alexa647 for 2 to 4 hours at 4°C. Subsequently, unbound dyes were removed by washing the column with 3 to 4 ml of buffer A. Elution of the proteins was done by supplementing buffer A with 200 mM imidazole. The labeled protein was further purified by size exclusion chromatography (Superdex 200, GE Healthcare) using buffer A. Sample composition was assessed by recording the absorbance at 280 nm (protein), 559 nm (Alexa555), and 645 nm (Alexa647) to estimate the labeling efficiency. The labeling efficiency was typically about 70%.
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