Fluorotect greenlys

Manufactured by Promega

FluoroTect GreenLys is a fluorescent reagent used to label and detect proteins in cell lysates. It binds to cysteine residues in proteins, producing a green fluorescent signal that can be measured using a fluorometer or fluorescence imager.

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Lab products found in correlation

Rnase a solution, by Promega (2 mentions) 1 step human coupled in vitro transcription translation kit, by Thermo Fisher Scientific (2 mentions) Synergy h1 plate reader, by Agilent Technologies (2 mentions) Purexpress kit, by New England Biolabs (2 mentions) Tnt coupled reticulocyte system, by Promega (2 mentions) Protein g magnetic beads, by Active Motif (1 mentions) Typhoon 9410, by GE Healthcare (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Mini protean tgx precast protein gel, by Bio-Rad (1 mentions) Fusion fx7 imaging system, by Vilber (1 mentions) Ni nta superflow resin, by Qiagen (1 mentions) Molecular dynamics typhoon 9410 molecular imager, by GE Healthcare (1 mentions) Transcend trna, by Promega (1 mentions) Ni nta bead, by Qiagen (1 mentions) Thermolysin, by Merck Group (1 mentions) Tnt sp6 high yield wheat germ protein expression system, by Promega (1 mentions) Gradient sds page gel, by Bio-Rad (1 mentions) Rnaseout, by Thermo Fisher Scientific (1 mentions) S30 t7 high yield protein expression system, by Promega (1 mentions) Purexpress, by New England Biolabs (1 mentions)

17 protocols using fluorotect greenlys

SDS-PAGE Analysis of Labeled Proteins

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PUREfrex2.0 reaction mixture was supplemented with 0.5 μL of GreenLys reagent (FluoroTect™ GreenLys, Promega) and gene expression was performed in a test tube as described above. Sample was treated with RNase (RNaseA Solution, Promega) for 30 min and proteins denatured for 10 min at 90 °C in 2× SDS loading buffer with 10 mM dithiotreitol (DTT). Samples were loaded on a 18% SDS-PAGE gel. Visualization of the fluorescently labeled protein was performed on a fluorescence gel imager (Typhoon, Amersham Biosciences) using a 488 nm laser and a band-pass emission filter of 520 nm.

Godino E., López J.N., Foschepoth D., Cleij C., Doerr A., Castellà C.F, & Danelon C. (2019). De novo synthesized Min proteins drive oscillatory liposome deformation and regulate FtsA-FtsZ cytoskeletal patterns. Nature Communications, 10, 4969.

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Immunoprecipitation of Yop Proteins

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Protein G magnetic beads (Active Motif, Carlsbad, CA) were coupled to 20 μL anti-YopD antibodies (non-purified rabbit sera, generously provided by Dr. Gregory Plano) in 250 μL 1% BSA in 1X PBS, pH 7.4 (Gibco) for 1 hr. at 4°C, with mixing. The coupled beads were collected and resuspended in the above BSA/PBS buffer. Soluble cell-free extracts with co-expressed Yop-NLPs and "empty"-NLPs (20 uL); and crude cell-free extracts with expressed Yop proteins (20 μL) in the presence of lipid (vesicle preparations) were added to the anti-YopD Protein G magnetic resuspended beads. The mixture was incubated at 4°C for 4 ½ hrs. with mixing. The beads were washed with 4–500 μL aliquots of 1X PBS, pH 7.4. Collected beads were then re-suspended in 25 μL 2X LDS and heat denatured. Samples (25 μL) were loaded on a 4–12% Bis-Tris gel and electrophoresed for 38 min. at 200V. The resulting gel was imaged for the incorporated Fluorotect green-Lys (Promega) using the green laser (532 nm) of a Typhoon 9410 (GE Healthcare) with a 526 nm bandpass filter.

Coleman M.A., Cappuccio J.A., Blanchette C.D., Gao T., Arroyo E.S., Hinz A.K., Bourguet F.A., Segelke B., Hoeprich P.D., Huser T., Laurence T.A., Motin V.L, & Chromy B.A. (2016). Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles. PLoS ONE, 11(3), e0150166.

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PURE Reaction RNAse Digestion Analysis

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PURE reactions (5 μL) labeled with FluoroTect GreenLys (Promega) were incubated with 0.8 μg or 0.2 μL of RNAse A solution (Promega) and incubated for 30 min at 37 °C and subsequently analyzed by SDS-PAGE using 10-well 4–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad). Gels were scanned (AlexaFluor 488 settings, excitation: Spectra blue 470 nm, emission: F-535 Y2 filter) with a Fusion FX7 Imaging System (Vilber) and analyzed with ImageJ. Protein sizes were calculated based on a BenchMark^TM Fluorescent Protein Standard (Invitrogen).

Lavickova B., Laohakunakorn N, & Maerkl S.J. (2020). A partially self-regenerating synthetic cell. Nature Communications, 11, 6340.

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Purification of Fluorescent Protein Fusions

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Two hundred μl of Superflow Ni-NTA resin (Qiagen) slurry was added to a 1 ml plastic column. Column was equilibrated with 1 ml native lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). Cell-free reaction mixture (ERBB2-NLP or YopD-NLP, pre-labeled with FluoroTect^™ GreenLys (Promega)) was added to the prepared Ni-NTA resin and mixed for 1 hour at 4C on a nutator. Columns were then washed with 2 × 200 μl of native lysis buffer with 10 mM imidazole, 2 × 200 μl of native lysis buffer with 20 mM imidazole. Samples were eluted with 2 × 200 μl of either 0.1 M sodium carbonate (pH 11.5), or native elution buffer, lysis buffer with 400 mM imidazole. Eluents were collected and concentrated using Vivaspin 500 spin column (MWCO = 10kD, Sartorius Stedim AG). The concentrated samples resolved by SDA-PAGE. Fluorescent pictures of the gels were taken using the blue laser (488 nm) of a Molecular Dynamics Typhoon 9410 Molecular Imager (GE Healthcare) with a filter 520 nm bandpass 30.

He W., Scharadin T.M., Saldana M., Gellner C., Hoang-Phou S., Takanishi C., Hura G.L., Tainer J.A., Carraway III K.L., Henderson P.T, & Coleman M.A. (2015). Cell-free expression of functional receptor tyrosine kinases. Scientific Reports, 5, 12896.

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Recombinant APOL1 Protein Expression

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His6-tagged APOL1 (E28-L398) or APOL1 variants were expressed from pStaby1.2 vector (Delphi Genetics) in E. coli after 4 h induction at 37 °C with 1 mM isopropyl β-D-thiogalactoside. After washing, inclusion bodies were dissolved in 6 M guanidium-HCl, 50 mM phosphate buffer (pH 8.0) and incubated with Ni-NTA beads (Qiagen) for 16 h at 4 °C. All washing steps occurred at pH 8. After elution and dialysis against 20 mM acetic acid, the protein was more than 96% pure, as determined by SDS–polyacrylamide gel electrophoresis. BODIPY Fl labelling was performed by simultaneous in vitro transcription and translation in a TnT Coupled Wheat Germ Extract System with either FluoroTect GreenLys or Transcend tRNA (Promega).

Vanwalleghem G., Fontaine F., Lecordier L., Tebabi P., Klewe K., Nolan D.P., Yamaryo-Botté Y., Botté C., Kremer A., Burkard G.S., Rassow J., Roditi I., Pérez-Morga D, & Pays E. (2015). Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1. Nature Communications, 6, 8078.

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Batch PURE Reactions for in vitro Protein Expression

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Batch PURE reactions (5 μL) were established by mixing 2 μL of 2.5× energy solution, 0.9 μL of 3.45 μM ribosomes (final concentration: 0.6 μM), 0.65 μL of PURE proteins (Supplementary Table 3), DNA template, and brought to a final volume of 5 μL with addition of water. All reactions measuring eGFP expression were prepared as described above with eGFP linear template at a final concentration of 4 nM and incubated at 37 °C at constant shaking for 3 h, and measured (excitation: 488 nm, emission: 507 nm) on a SynergyMX platereader (BioTek). The eGFP production rate was calculated between 20 and 50 min based on an eGFP calibration curve (Supplementary Fig. 24a). Reactions expressing other proteins were prepared as described above and supplemented with 0.2 μL FluoroTect GreenLys (Promega). DNA templates were used at a final concentration of 2 nM and the reactions were incubated at 37 °C for 3 h.

Lavickova B., Laohakunakorn N, & Maerkl S.J. (2020). A partially self-regenerating synthetic cell. Nature Communications, 11, 6340.

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In Vitro Transcription Monitoring

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IVT reactions were performed as per the manufacturer’s instructions but with the addition of 1 μl of RNase inhibitor, murine, and 1.25 μl of FluoroTect GreenLys (Promega) per 30 μl of IVT reaction and initiated with 250 ng of DNA. At each time point, 1.5 μl of IVT reaction was quenched into a final concentration of 2 mM chloramphenicol and RNase A (0.1 mg/ml) and then SDS-PAGE loading dye. Reactions were then run on a 4 to 12% bis-tris gel in an MES run buffer and imaged using a Typhoon Trio. Analysis was performed using ImageJ.

Samelson A.J., Bolin E., Costello S.M., Sharma A.K., O’Brien E.P, & Marqusee S. (2018). Kinetic and structural comparison of a protein’s cotranslational folding and refolding pathways. Science Advances, 4(5), eaas9098.

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In Vitro Transcription and Refolding

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IVT reactions were performed as per the manufacturer’s instructions but with the addition of 1 μl of RNase inhibitor, murine, and 1.25 μl of FluoroTect GreenLys (Promega) per 30 μl of IVT reaction (25 ). IVT reactions were quenched after 1 hour to a final concentration of 2 mM chloramphenicol and RNase A (0.1 mg/ml). Refolding experiments were performed as described above. IVT reactions and refolding reactions were allowed to reach equilibrium for at least 12 hours. Subsequently, reactions were aliquoted to 10 μl, and 1 μl of thermolysin (1 mg/ml; Sigma) was added to each reaction for 1 min and quenched with EDTA to a final concentration of 83 mM. SDS-PAGE loading dye was then added to each reaction, and each reaction was run on a 4 to 12% bis-tris gel in an MES run buffer. Imaging and analysis was performed as described previously (28 (link)).

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In Vitro Protein Production via Wheat Germ System

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Plasmids were used to produce proteins through coupled in vitro transcription-translation, using TnT SP6 High-Yield Wheat Germ Protein Expression System (Promega), as per the manufacturer's instructions. Protein synthesis was initiated by mixing the appropriate DNA template (2–3 µg), 30 µL of the TnT SP6 High-Yield Wheat Germ Master Mix and water for a 50 µL final volume, and then incubating the reaction at 25°C for 2 hours. Protein expression was analyzed by the incorporation of labelled lysine residues (FluoroTect GreenLys, Promega) in a 10 µl aliquot of the plasmid/wheat germ lysate mixture (WGL) as directed in the instructions. Samples were heated for 3 minutes at 70°C, run on 4–20% SDS-PAGE gradient gels (BioRad, UK), under reducing conditions and imaged with a laser-based fluorescent gel scanner (Fujifilm LAS-4000 319 Imaging System). Molecular weights (MW were estimated using the Kaleidoscope Precision plus marker (BioRad, UK) which contains several fluorescently labelled components.

Wan D., Ludolf F., Alanine D.G., Stretton O., Ali Ali E., Al-Barwary N., Wang X., Doenhoff M.J., Mari A., Fitzsimmons C.M., Dunne D.W., Nakamura R., Oliveira G.C., Alcocer M.J, & Falcone F.H. (2014). Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins. PLoS Neglected Tropical Diseases, 8(9), e3124.

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In Vitro Protein Expression and Characterization

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Thermo Fisher 1-step human coupled in vitro transcription-translation kit (cat# 88881) was used to synthesize proteins expressed under the T7 promoter. To express the proteins, the protocol provided by the manufacturer was followed. In short, 5 μL of HeLa lysate was mixed with 2 μL reaction mixture and 1 μL accessory proteins. The reaction volume was then brought to 10 μL by adding 10 nM DNA plasmid and ultra-pure water. In cases where direct reconstitution of InterTag or InterCatch was desired, water was replaced by 5 mM 100% DOPC SUV solution so that the final concentration of SUV in the reaction was around 1 mM. The reactions were next transferred to a 384 conical well plate and incubated in the Synergy H1 plate reader (BioTek) at 30 °C for 3–4 h. The GFP, BFP, and sfCherry signals were monitored using plate readers at 400/450 nm, 488/528 nm, and 561/625 nm excitation/emission wavelengths, respectively. For experiments that required in vitro translation labeling system, a 1:50 dilution of FluoroTect^™ GreenLys (Promega) was added to the reaction before incubation.

Moghimianavval H., Patel C., Mohapatra S., Hwang S.W., Kayikcioglu T., Bashirzadeh Y., Liu A.P, & Ha T. (2022). Engineering functional membrane-membrane interface by InterSpy. Small (Weinheim an der Bergstrasse, Germany), 19(13), e2202104.

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