Tnt coupled wheat germ extract system

Manufactured by Promega

Sourced in United States

The TNT Coupled Wheat Germ Extract System is a cell-free protein expression system that allows for the in vitro synthesis of proteins from DNA templates. The system utilizes an extract derived from wheat germ, which contains the necessary cellular machinery to transcribe and translate DNA into functional proteins.

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Lab products found in correlation

Immobilon psq membrane, by Merck Group (3 mentions) 35s methionine, by Hartmann Analytic (3 mentions) Novex tricine sds sample buffer 2x, by Thermo Fisher Scientific (3 mentions) Flag ha modified pcdna3.1 vector, by Thermo Fisher Scientific (2 mentions) Ha affinity gel beads, by Thermo Fisher Scientific (2 mentions) Pgex 6p 1 vector, by GE Healthcare (2 mentions) Magne halotag beads, by Promega (2 mentions) Lds sample buffer, by Thermo Fisher Scientific (1 mentions) Anti myc antibody, by Merck Group (1 mentions) Gsh sepharose beads, by GE Healthcare (1 mentions) Anti ha antibody 3f10, by Roche (1 mentions) Magnegst pull down system, by Promega (1 mentions) Tricine gel, by Thermo Fisher Scientific (1 mentions) Ubiquitin aldehyde, by R&D Systems (1 mentions) Itch gst, by Abnova (1 mentions) Ubch7, by R&D Systems (1 mentions) Membrane lipid strip, by Echelon Biosciences (1 mentions) Gst spintrap column, by GE Healthcare (1 mentions) Spintrap, by Cytiva (1 mentions) Tnt coupled reticulocyte lysate system, by Promega (1 mentions)

Spelling variants of this product

TNT systems (37 citations) Wheat germ extract (20 citations) TNT SP6 Coupled Wheat Germ Extract System (16 citations) TNT T7 Coupled Wheat Germ Extract System (13 citations) Wheat germ extract system (7 citations) TNT T7/SP6 Coupled Wheat Germ Extract System (2 citations)

19 protocols using tnt coupled wheat germ extract system

In Vitro Transcription and O-GlcNAcylation Assay

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HCF-1 was in vitro transcribed and translated using the S6 promoter of the pCMV-Sport6 HCF-1 plasmid and the TnT® Coupled Wheat Germ Extract System per manufacturer’s instructions (Promega, L4130).
His-OGT WT or OGT D554N proteins were purified from E. Coli as previously described (Janetzko et al., 2016 (link)), and used to process the in vitro translated HCF-1. Briefly, 5 μl of the in vitro synthesized HCF-1 protein was incubated with 35 μl of in vitro cleavage and O-GlcNAcylation assay buffer (20 mM Tris/HCl pH 7.5, 120 mM NaCl, 2 mM MgCl, 2 mM UDP-GlcNAc, 1mM fresh DTT) containing 1 or 2 μg of His-OGT WT or OGT D554N for 3 hrs at 37°C.
For interaction assays, endogenous ChREBP was bound to protein G agarose beads as described in the immunoprecipitation method above. 30 μl of pre-washed, ChREBP bound beads was incubated with 40 μl of the in vitro processed HCF-1 protein that was diluted in 110 μl of Co-IP lysis buffer (20 mM Tris/HCl pH 7.5, 137 mM NaCl, 5% glycerol, 2 mM EDTA, 2 mM Na₃VO₄, Complete™ EDTA-free Protease Inhibitor Cocktail, Phosphatase inhibitor cocktail 3, 2 μM Thiamet-G, 10 μM PUGNAc, 1 μM TSA, 0.15% Triton-X100). After 1 hr rotation at 4°C, the complex was washed 2 times with lysis buffer, eluted in 50 μl of 2X LDS sample buffer (Life Technologies) and boiled for 15 min for western blot analysis.

Lane E.A., Choi D.W., Garcia-Haro L., Levine Z.G., Tedoldi M., Walker S, & Danial N.N. (2019). HCF-1 regulates de novo lipogenesis through a nutrient sensitive complex with ChREBP. Molecular cell, 75(2), 357-371.e7.

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ARID1 Protein Binding Assay

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DNA binding assays were performed as described [41] (link) with the following modifications. Briefly, biotinylated DNA fragments corresponding to 3, 4, 5, and 10 (Figure 3) were generated by PCR using primer pairs DUO1_F3/R3, DUO1_F4/R4, DUO1_F5/R5, and DUO1_F10/R10, with labeling by 5′biotin at F3, F4, F5, and F10, respectively. Then 100 pmol of the biotinylated DNA fragments were incubated with 50 ul prewashed Streptavidin Agoraose Resin (Thermo, Cat.20349) in IP100 buffer (100 mM potassium glutamate, 50 mM Tris-HCl pH 7.6, 2 mM MgCl2, 0.5% NP40) for 2 h at room temperature with slight rotation, and washed five times in IP100 buffer. In parallel, ARID1 was subjected to TNT T7 in vitro transcription/translation with the TNT Coupled Wheat Germ Extract System (Promega, Cat.L4140). 25 ul freshly translated ARID1 protein was added to DNA-bound beads in the IP buffer plus complete protease inhibitor cocktail, and the mixture was rotated at 4°C for 2 h. Beads were washed eight times with IP100 buffer, then proteins were stripped off the beads by boiling with 2XSDS buffer and then subjected to SDS-PAGE. The ARID1 protein bound by biotinylated DNA was detected by immunoblotting with a 1∶200 dilution of anti-ARID1 (The peptide “SMVADEDAVDYSKT” was conjugated to KLH and used to raise rabbit polyclonal antibodies (GL Biochem)).

Zheng B., He H., Zheng Y., Wu W, & McCormick S. (2014). An ARID Domain-Containing Protein within Nuclear Bodies Is Required for Sperm Cell Formation in Arabidopsis thaliana. PLoS Genetics, 10(7), e1004421.

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Quantification of ASK1 Protein Interactions

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Five µg of GST-ASK1 protein or its mutated versions were immobilized in GSH-sepharose beads (GE Healthcare, USA), and incubated with 100 µM NO-Cys in 200 μl of 1X PBS buffer during 1 h in the dark. DTT was added for 10 min as control. After washing samples with 10 bed volumes of 1X PBS, beads were incubated with TIR1-myc during 30 min. TIR1-myc was obtained by in vitro translation using TNT coupled wheat germ extract system (Promega, USA) according to Terrile et al. [80] (link). Finally, proteins were eluted in 50 mM Tris-HCl pH 8.0 containing 200 mM NaCl and 10 mM GSH, denatured and separated on 15% SDS-PAGE. TIR1-myc was detected by immunoblotting with anti-myc antibody (Sigma-Aldrich, USA).

Iglesias M.J., Terrile M.C., Correa-Aragunde N., Colman S.L., Izquierdo-Álvarez A., Fiol D.F., París R., Sánchez-López N., Marina A., Calderón Villalobos L.I., Estelle M., Lamattina L., Martínez-Ruiz A, & Casalongué C.A. (2018). Regulation of SCFTIR1/AFBs E3 ligase assembly by S-nitrosylation of Arabidopsis SKP1-like1 impacts on auxin signaling. Redox Biology, 18, 200-210.

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Protein-Protein Interaction Assay Using GST-Tagged Constructs

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Pull-down assays were carried out using the MagneGST^TM Pull-Down System (Promega, Madison, WI) by combining human influenza hemagglutinin (HA)-tagged and glutathione S-transferase (GST) fusion proteins. In vitro transcription/translation of HA-tagged ORO proteins was carried out using the TNT Coupled Wheat Germ Extract System (Promega, Madison, WI). GST-tagged SAM proteins were expressed in Escherichia coli. Protein production was induced by adding IPTG to a final concentration of 2 mM and shaking for 20 hr at 16°C. After the capture phase, beads were washed four times with 400 μL of washing buffer (0.5% IGEPAL, 290 mM NaCl, 10 mM KCl, 4.2 mM Na₂HPO₄, 2 mM KH₂PO₄, at pH 7.2) at room temperature. Beads were then recovered in SDS-PAGE loading buffer, and proteins analysed by SDS-PAGE followed by Clarity^TM chemiluminescent detection (Biorad, Hercules, CA). The anti-HA antibody (3F10) was purchased from Roche, and the anti-GST antibody (91G1) from Ozyme.

Arun A., Coelho S.M., Peters A.F., Bourdareau S., Pérès L., Scornet D., Strittmatter M., Lipinska A.P., Yao H., Godfroy O., Montecinos G.J., Avia K., Macaisne N., Troadec C., Bendahmane A, & Cock J.M. (2019). Convergent recruitment of TALE homeodomain life cycle regulators to direct sporophyte development in land plants and brown algae. eLife, 8, e43101.

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Purification and Binding of Phf5a Protein

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Phf5a ORF was cloned into the pGEX-6P-1 vector (GE Healthcare) (kind gift from Dr. K.J. Armache, NYU School of Medicine) and transformed into BL21 Star (DE3) bacteria. 12L of liquid cultures were induced overnight with 0.1mM IPTG at 18°C. Bacteria were lysed in 20mM Tris-HCl pH8.0, 200mM NaCl, 1mM DTT passing though a pressure homogenizer. Soluble fraction was bound to glutathione agarose beads (Pierce) for 1h at 4°C, beads were washed with 200 column volumes (CV) Lysis Buffer and GST-Phf5a protein was eluted using reduced glutathione. GST tag was cleaved by cleavage with PreScission Protease (kind gift from Dr. K.J. Armache, NYU School of Medicine) and Phf5a was further purified by ion exchange and size exclusion FPLC chromatography. Paf1, Cdc73 and Wdr61 were cloned in Flag/HA-modified pCDNA3.1 vector (Invitrogen) and in vitro translated using TNT Coupled Wheat Germ Extract System (L4140, Promega). 5µg of purified Phf5a protein was added, mixtures were bound overnight at 4°C and HA-tagged proteins were pulled-down using HA affinity gel beads (Invitrogen) for 4h at 4°C. Beads were washed 4 times with 1mL Lysis Buffer and interactions were visualized by WB analysis.

Strikoudis A., Lazaris C., Trimarchi T., Neto A.L., Yang Y., Ntziachristos P., Rothbart S., Buckley S., Dolgalev I., Stadtfeld M., Strahl B.D., Dynlacht B.D., Tsirigos A, & Aifantis I. (2016). Regulation of transcriptional elongation in pluripotency and cell differentiation by the PHD-finger protein Phf5a. Nature cell biology, 18(11), 1127-1138.

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In Vitro Synthesis of LINC01013 Protein

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The full LINC01013 RNA sequence (including 5′UTR, ORF region and 3′UTR) was synthesized and inserted into an expression plasmid by Genewiz Europe (Leipzig, Germany; construct available upon request). Linearized plasmid DNA (0.5 µg) was transcribed and translated in vitro as described previously [25 ] using the TnT Coupled Wheat Germ Extract system (Promega, Mannheim, Germany) in the presence of 10 mCi/mL [35S]-methionine (Hartmann Analytic, Braunschweig, Germany) according to manufacturer’s instructions. To visualise the translation products, 5µL lysate was denatured for 2 min at 85 °C in 9.6 µL Novex Tricine SDS Sample Buffer (2X) (Thermo Fisher Scientific) and 1.4 µL DTT (500 mM). Proteins were separated on 16% Tricine gels (Invitrogen) for 1 h at 50 V followed by 3.5 h at 100 V and blotted on PVDF-membranes (Immobilon-PSQ Membrane, Merck Millipore).

Quaife N.M., Chothani S., Schulz J.F., Lindberg E.L., Vanezis K., Adami E., O’Fee K., Greiner J., Litviňuková M., van Heesch S., Whiffin N., Hubner N., Schafer S., Rackham O., Cook S.A, & Barton P.J. (2022). LINC01013 Is a Determinant of Fibroblast Activation and Encodes a Novel Fibroblast-Activating Micropeptide. Journal of Cardiovascular Translational Research, 16(1), 77-85.

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In vitro Ubiquitylation of SuFu Protein

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In vitro ubiquitylation was performed as previously described^{64 (link),65 (link)}. In vitro translated protein SuFu, produced using TnT^® Coupled Wheat Germ Extract System (Promega, Madison, WI, USA), was incubated at 30 °C with GST or Itch-GST or Itch-GST and βarr2-GST 400ng (Abnova, Heidelberg, Germany), 50 mM Tris-HCl at pH 7.5, 5 mM MgCl₂, 200 µM okadaic acid, 2 mM ATP, 0.6 mM DTT, 1 mM ubiquitin aldehyde, E1, UbcH7, and either wild-type or mutant ubiquitin (Boston Biochem). Polyubiquitylated products were subjected to SDS-PAGE and detected by fluorography.

Infante P., Faedda R., Bernardi F., Bufalieri F., Lospinoso Severini L., Alfonsi R., Mazzà D., Siler M., Coni S., Po A., Petroni M., Ferretti E., Mori M., De Smaele E., Canettieri G., Capalbo C., Maroder M., Screpanti I., Kool M., Pfister S.M., Guardavaccaro D., Gulino A, & Di Marcotullio L. (2018). Itch/β-arrestin2-dependent non-proteolytic ubiquitylation of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis. Nature Communications, 9, 976.

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CIDEA-v5 Binding to Phosphatidic Acid

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In vitro translated CIDEA-v5 was synthesized from pcDNA3.1/Cidea-v5 using the TnT Coupled Wheat Germ Extract System (Promega) and verified by Western blot. The cell-free preparation of CIDEA-v5 was probed with Membrane Lipid Strips (Echelon Biosciences) following the manufacturer’s instructions. Protein affinity for PA was examined in pull-down assays using PA covalently linked to agarose beads (PA beads) (Manifava et al., 2001 (link)). Cells were lysed in 50 mM Tris-HCl pH 8.0, 50 mM KCl, 10 mM EDTA, 0.5% Nonidet P-40, and protease inhibitors. Lysates were sonicated and centrifuged at 14000g prior to incubation with the PA beads as previously described (Manifava et al., 2001 (link)). Competition experiments with soluble phospholipids were performed by supplementing the cell lysate with 1,2-dilauroyl-sn-glycero-3-phosphate 12:0 PC (DLPA) or 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) (Echelon Biosciences). Each PA-binding experiment was performed at least in triplicate, producing similar results in each experiment with a representative image presented.

Barneda D., Planas-Iglesias J., Gaspar M.L., Mohammadyani D., Prasannan S., Dormann D., Han G.S., Jesch S.A., Carman G.M., Kagan V., Parker M.G., Ktistakis N.T., Klein-Seetharaman J., Dixon A.M., Henry S.A, & Christian M. (2015). The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix. eLife, 4, e07485.

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Recombinant Cdh1 and Borealin Protein Production

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To prepare in vitro translated (IVT) Cdh1 recombinant protein, the TNT Coupled Wheat Germ Extract System (Promega) was used with pcDNA3.1-Cdh1 vector, following the manufacturer's protocol. For GST-tagged recombinant protein production, pGEX-4T-1-borealin vector was transformed to BL21(DE3) (Nippon gene) and GST-tagged borealin was expressed for 15 h at 18°C with 0.2 mM isopropyl-1-thio-D-galactopyranoside (IPTG). Cells were lysed using NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl pH 7.5, 1 mM PMSF, 5 mM benzamidine and 0.5% NP-40) and sonicated. After centrifugation, cleared lysate was collected and incubated in GST SpinTrap columns (GE healthcare) for 1 h at 4°C. Then, each column was washed three times with binding buffer (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.5 mM EDTA, 10 mM NaF, 0.1% Nonidet P-40, 20 mM β-glycerophosphate and 20 nM okadic acid) and incubated with 15 μl IVT Cdh1 and binding buffer for 2 h at 4°C. After incubation, each column was washed five times with binding buffer and proteins were eluted using GST elution buffer (GE healthcare). The eluted proteins were analyzed using SDS–PAGE.

Tsunematsu T., Arakaki R., Kawai H., Ruppert J., Tsuneyama K., Ishimaru N., Earnshaw W.C., Pagano M, & Kudo Y. (2020). APC/CCdh1 is required for the termination of chromosomal passenger complex activity upon mitotic exit. Journal of Cell Science, 133(18), jcs251314.

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Transcription factor binding analysis

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pCS2+ plasmid expression vectors for Olig2, E12, Ngn2, and Id1 [13 (link)] were transcribed and translated in vitro using the Promega TNT Coupled Wheat Germ Extract System. Programmed extracts were mixed as indicated in a buffer containing 100 mM Hepes pH 7.6, 25 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 2.5% glycerol, and 100 ng poly dIdC and incubated for 15 minutes at room temperature. ³²P-dCTP-labeled probes were generated by Klenow end labeling of double-stranded oligonucleotides containing the native mouse Hes5(e1) sequence (forward 5′- ggccgCTCCCAAAAGACCATCTGGCTCCGTGTTATAA-3′; reverse 5′- actagTTATAACACGGAGCCAGATGGTCTTTTGGGAG-3′) or an E-box mutated version (forward 5′ ggccgCTCCCAAAAGAggATCccGCTCCGTGTTATAA-3′; reverse 5′-actagTTATAACACGGAGCggGATccTCTTTTGGGAG-3′). The E-box sequence is underlined. Lowercase indicates substitutions and added flanking sequences. Samples were incubated with labeled probes for 15 minutes before resolving on a 4.5% polyacrylamide gel and subsequent autoradiography. Probe competition was achieved by incorporating unlabeled oligonucleotide probes in the binding reaction mix.

Sagner A., Gaber Z.B., Delile J., Kong J.H., Rousso D.L., Pearson C.A., Weicksel S.E., Melchionda M., Mousavy Gharavy S.N., Briscoe J, & Novitch B.G. (2018). Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics. PLoS Biology, 16(2), e2003127.

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