Fluorotect greenlys in vitro translation labeling system

Manufactured by Promega

Sourced in United States

The FluoroTect™ GreenLys in vitro Translation Labeling System is a tool used to detect and quantify newly synthesized proteins. It incorporates a fluorescent amino acid analog that is incorporated into proteins during in vitro translation, enabling the visualization and analysis of protein expression.

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Lab products found in correlation

Tnt quick coupled transcription translation systems kit, by Promega (1 mentions) Purexpress kit, by New England Biolabs (1 mentions) Luciferase t7 control dna, by Promega (1 mentions) Tnt t7 coupled wheat germ extract system, by Promega (1 mentions) Precision protease, by GE Healthcare (1 mentions) Tnt sp6 high yield wheat germ protein expression system, by Promega (1 mentions) Glutathione sepharose 4b, by GE Healthcare (1 mentions) Reducing agent, by Thermo Fisher Scientific (1 mentions) Seeblue plus2, by Thermo Fisher Scientific (1 mentions) Sypro ruby protein gel stain, by Bio-Rad (1 mentions) Lds sample buffer, by Thermo Fisher Scientific (1 mentions) Typhoon 9410, by GE Healthcare (1 mentions) Bis tris gel, by Thermo Fisher Scientific (1 mentions)

7 protocols using fluorotect greenlys in vitro translation labeling system

TRPM8 Binding Interactions with Rap1 Proteins

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TRPM8 N- and C-terminal tail GST-fusion proteins were produced and purified as described previously (Gkika et al., 2015 (link)). GST-fused purified proteins were then incubated with HEK cell lysates transfected with pEGFP-RAP-1, pEGFP-RAP-1N17, or pEGFP-RAP-1V12 plasmids (Bivona et al., 2004 (link)). For the direct interaction assay, the coding sequence of RAP-1-WT, RAP N17, and RAP V12 were subcloned in the pCMV TNT vectors (Promega) as EcoR1–Not1 fragments to produce them in vitro. Subsequently, Rap-WT and mutant proteins were translated in vitro using the TNT Quick Coupled Transcription/Translation Systems kit (Promega) and the FluoroTect GreenLys in vitro Translation Labeling System (Promega) as per the manufacturer’s instructions. For GST pull-down experiments performed with Rap1-WT loaded with GDP and GTP, in vitro translated Rap1-WT was incubated with 10 mM EDTA followed by 1 mM GDP or 100 μM GTPγS for 30 min at 30°C with constant agitation. The sample was then placed on ice, 60 mM MgCl₂ was added, and the sample was vortexed.
Cell lysates or in vitro translated proteins were incubated overnight at 4°C together with the purified GST-fusion proteins. Subsequently, beads were washed extensively and bound proteins were eluted with SDS-PAGE loading buffer, separated on 4–20% wt/vol SDS-PAGE gels, and visualized by fluorescence imaging (Bio-Imager 600; GE Healthcare).

Genova T., Grolez G.P., Camillo C., Bernardini M., Bokhobza A., Richard E., Scianna M., Lemonnier L., Valdembri D., Munaron L., Philips M.R., Mattot V., Serini G., Prevarskaya N., Gkika D, & Pla A.F. (2017). TRPM8 inhibits endothelial cell migration via a non-channel function by trapping the small GTPase Rap1. The Journal of Cell Biology, 216(7), 2107-2130.

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In vitro Synthesis and Characterization of AatR2

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The DNA template for in vitro transcription-translation of ataR2 was prepared by PCR using the pBAD-ataRT2 vector template and T7-ataR2-F and T7-ataR2-R primers. The in vitro coupled transcription–translation reactions were carried out by PURExpress kit (NEB, USA) using DNA templates encoding ataR2 or the dihydrofolate reductase (DHFR) (NEB, USA). The in vitro translation of firefly luciferase was carried our with in vitro transcribed mRNA (Luciferase T7 Control DNA Promega, USA). Before template addition, translation reactions were incubated for 10 min at 37°C in the presence of 0.2 mM of acetyl coenzyme A, with and without 2 μM AtaT2. DHFR- and ataR2- translation reaction mixtures were supplemented with FluoroTect™ GreenLys in vitro Translation Labeling System (Promega, USA). The fluorescent products of in vitro translation reactions were analyzed by 12% SDS-PAGE, and visualized by Typhoon fluorescence gel imager (GE, USA). The enzymatic activity of in vitro synthesized luciferase was measured by detection of chemiluminescence in the presence of 0.1 mM d-luciferin with VictorX5 multireader (Perkin-Elmer, USA).
The toeprinting assay was carried out using Rst1 and Rst2 mRNA as templates as described in (25 (link)), except that reactions were preincubated for 10 min with 0.4 mM Ac-CoA with or without the addition of 2 μM AtaT2.

Ovchinnikov S.V., Bikmetov D., Livenskyi A., Serebryakova M., Wilcox B., Mangano K., Shiriaev D.I., Osterman I.A., Sergiev P.V., Borukhov S., Vazquez-Laslop N., Mankin A.S., Severinov K, & Dubiley S. (2020). Mechanism of translation inhibition by type II GNAT toxin AtaT2. Nucleic Acids Research, 48(15), 8617-8625.

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Granzyme B Protease Activity Assay

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Recombinant Glosensor proteins were generated using the TNT T7 Coupled Wheat Germ Extract System (Promega) supplemented with the FluoroTect GreenLys in vitro Translation Labeling System (Promega). Following cell-free expression for two hours at 30 °C, aliquots of cell-free expression lysate were combined with an equal volume of murine granzyme B (Sigma) in assay buffer (100 mM HEPES, pH 7.4, 200 mM NaCl, 0.2% CHAPS, 2 mM EDTA, 20% glycerol). Following a one hour incubation at 37 °C, 10 μL of sample was combined with 90 μL of Luciferase Assay Reagent and luminescence was measured at room temperature on a GloMax Multi+ luminometer (0.5 second integration time).

Li J., Figueira S.K., Vrazo A.C., Binkowski B.F., Butler B.L., Tabata Y., Filipovich A., Jordan M.B, & Risma K.A. (2014). Real time detection of cytotoxic lymphocyte function reveals distinct patterns of caspase activation mediated by Fas versus granzyme B. Journal of immunology (Baltimore, Md. : 1950), 193(2), 519-528.

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SKIK-fused Zipbody Expression in E. coli

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To express SKIK-fused Zipbody, the DNA templates of Hc and Lc were amplified from the above assembled vectors with primers annealed to upstream of the T7 promoter and downstream of the T7 terminator. E. coli-based PURE system (GeneFrontier, Kashiwa, Japan) with oxidized glutathione (GSSG) and DsbC were used as recommended by the manufacturer. Proteins were expressed on a 10-µL scale containing 0.5 µL PCR product or 20 ng purified DNA fragments (each of Hc and Lc) as the template, at 37 °C for 90 min. FluoroTect™ Green_Lys in vitro Translation Labeling System (Promega, Madison, WI, USA) was included to confirm protein synthesis in subsequent SDS-PAGE analysis.

Ojima-Kato T., Morishita S., Uchida Y., Nagai S., Kojima T, & Nakano H. (2018). Rapid Generation of Monoclonal Antibodies from Single B Cells by Ecobody Technology. Antibodies, 7(4), 38.

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Expression and Purification of Recombinant ASC Proteins

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BL21 (DE3) cells were transformed with the pGEX-ASC expression vectors for full-length ASC or deletion mutants and were cultured in LB with ampicillin at 37°C until an optical density at 600 nm (OD₆₀₀ nm) of 0.8 was reached. Protein expression was induced by the addition of 1 mM isopropyl-β-thiogalactopyranoside at 37°C for 4 hours. Cell pellets were resuspended in buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.1% Tween-20, sonicated twice for 5 minutes at 4°C, and centrifuged at 20,000g for 30 minutes. The supernatant was incubated with Glutathione Sepharose 4B (GE Healthcare, cat. no. 17075601) at 4°C for 1 hour. Then, the resin was washed five times with the above buffer, the GST tag was cleaved by the addition of Precision Protease (GE Healthcare, cat. no. 27084301), and the sample was incubated at 4°C for another 16 hours. Cleaved proteins were recovered from the supernatants and stored at –80°C.
For the preparation of in vitro–translated proteins, vectors expressing NLRP1, 3, 4, or 6 under the control of an SP6 promoter (5 μg) were incubated with 50 μL of TNT SP6 High-Yield Wheat Germ Protein Expression System (Promega, cat. no. L3260) and fluorescently labeled using FluoroTect Green_Lys in vitro Translation Labeling System (Promega, cat. no. L5001) according to the manufacturer’s instructions.

Tsugawa H., Kabe Y., Kanai A., Sugiura Y., Hida S., Taniguchi S., Takahashi T., Matsui H., Yasukawa Z., Itou H., Takubo K., Suzuki H., Honda K., Handa H, & Suematsu M. (2020). Short-chain fatty acids bind to apoptosis-associated speck-like protein to activate inflammasome complex to prevent Salmonella infection. PLoS Biology, 18(9), e3000813.

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Cell-free Protein Expression Using E. coli PURE

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The reconstituted E. coli-based CFPS PURE system (GeneFrontier, Kashiwa, Japan), with or without DsbC, was used. Oxidised glutathione (GSSG) was also included when DsbC was added to the reaction mixture, as recommended by the manufacturer. Proteins were expressed on a 10-µL scale containing 0.5 µL PCR product or 20 ng purified DNA fragments (each of Hc and Lc) as the template, at 37 °C for 90 min, using a thermal cycler. FluoroTect™ GreenLys in vitro Translation Labeling System (Promega, Madison, WI) was included to confirm protein synthesis in subsequent SDS-PAGE analysis, as described previously^{17 (link)}.

Ojima-Kato T., Nagai S, & Nakano H. (2017). Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation. Scientific Reports, 7, 13979.

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SDS-PAGE Analysis of Yop Proteins

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For small scale reactions, a 1 μL aliquot of the total (T) cell-free reaction, soluble (S) fraction and resuspended pellet (I) were diluted with 1x LDS Sample buffer with reducing agents (Invitrogen), heat denatured and loaded on to a 4–12% gradient pre-made Bis-Tris gel (Invitrogen) along with the molecular weight standard SeeBlue plus2 (Invitrogen). The running buffer was 1X MES-SDS (Invitrogen). Samples were electrophoresed for 38 minutes at 200V. Gels were imaged using the green laser (532 nm) of a Typhoon 9410 (GE Healthcare) with a 526 nm bandpass 30 filter for the detection of the produced Yop proteins and/or apolipoprotein with incorporated fluorescently labeled lysine (FluoroTect™ GreenLys in vitro Translation Labeling System, Promega), gels were subsequently stained with coomassie brilliant blue or SYPRO Ruby Protein Gel Stain (Bio-Rad) (Data not shown).

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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