Tnt coupled transcription and translation system

Manufactured by Promega

Sourced in United States

The TNT-coupled transcription and translation system is a lab equipment product that facilitates the in vitro synthesis of proteins from DNA templates. It provides the necessary components for the simultaneous execution of transcription and translation processes, allowing for the direct production of proteins.

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Lab products found in correlation

Glutathione sepharose 4b, by GE Healthcare (2 mentions) Glutathione sepharose 4b bead, by Cytiva (1 mentions) Ubiquitin, by Cell Signaling Technology (1 mentions) C ebpα, by Cell Signaling Technology (1 mentions) Gst sepharose beads, by Cytiva (1 mentions)

Close spelling variants of this product

TNT systems (37 citations) TNT Quick Coupled Transcription and Translation System (6 citations)

7 protocols using tnt coupled transcription and translation system

GST pull-down assay for protein-protein interactions

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pcDNA3.1 constructs with Flag tag were translated
in vitro by using the TNT-coupled transcription and translation system (Promega, Madison, USA) following the manufacturer’s instruction. GST constructs were transformed into
Escherichia coli BL21 cells and the protein expression was induced by IPTG (up to 1 mM) at 37°C for 4 h. The GST or GST fusion proteins were extracted and immobilized onto Glutathione Sepharose 4B beads (Amersham Biosciences, Piscataway, USA) at 4°C for 4 h. Equal amounts of GST and GST-fusion proteins were incubated with
in vitro translated protein at 4°C overnight. After centrifugation and three times wash with lysis buffer, the beads were eluted with 30 μL of 1×SDS loading buffer and then boiled for 10 min, followed by western blot analysis using anti-Flag-tag M2 antibody (1:5000) as the primary antibody. Ponceau stain indicated the loading of GST or GST-fusion proteins.

Li X., Chen M., Yuan Y., Li J, & Li F. (2022). CORO1C, a novel PAK4 binding protein, recruitsphospho-PAK4 at serine 99 to the leading edge and promotes the migration of gastric cancer cells: CORO1C recruits PAK4 to the membrane of gastric cancer cells. Acta Biochimica et Biophysica Sinica, 54(5), 673-685.

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In Vitro Protein Expression and Analysis

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In vitro transcription and translation of His-MORC2 or Flag-C/EBPα proteins were performed by using the TNT-coupled transcription and translation system (Promega), as previously described [20 (link)]. The procedures of western blot and Immunoprecipitation assays were performed as previously described [17 (link)]. Primary antibody dilution and order company of His (1:2000, GenScript Corporation), c-myc and sumo1 (1:1000, Santa Cruz) and C/EBPα and Ubiquitin(1:1000, Cell Signaling Technology); Flag (1:2000, Shang Hai, Genomics); MORC2 (1:2000, Abcam);TFF1(1:1000, Protein tech Group).

Liu J., Zhang Q., Ruan B., Chen W., Zheng J., Xu B., Jiang P., Miao Z., Li F., Guo J.Y., Cao L, & Wang G. (2019). MORC2 regulates C/EBPα-mediated cell differentiation via sumoylation. Cell Death and Differentiation, 26(10), 1905-1917.

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HA-Alox5 Binding to GST-Runx2 In Vitro

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The TNT coupled transcription and translation system (Promega) was used to transcribe and translate HA-Alox5 protein in vitro. Prokaryotically expressed GST-Runx2 in HEK 293T was purified with glutathione Sepharose 4B (GE Healthcare). HA-Alox5 protein and GST-Runx2 were co-incubated and rotated in 1% NP-40 buffer at 4 °C for 3 h. Subsequently, the protein complex was washed three times, eluted with 30 μl of 2 × SDS loading buffer and analyzed with Western Blotting assay.

Xie S., Chen M., Fang W., Liu S., Wu Q., Liu C., Xing Y., Shi W., Xu M., Zhang M., Chen S., Zeng X., Wang S., Deng W, & Tang Q. (2022). Diminished arachidonate 5-lipoxygenase perturbs phase separation and transcriptional response of Runx2 to reverse pathological ventricular remodeling. eBioMedicine, 86, 104359.

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In Vitro p53-ATF2 Protein Interaction

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The TNT coupled transcription and translation system (Promega) was used to transcribe and translate Flag-p53 protein in vitro. Prokaryotically expressed GST-ATF2 in BL21 bacteria strain was purified with glutathione Sepharose 4B (GE Healthcare). Flag-p53 protein and GST-ATF2 were co-incubated and rotated in 1% NP-40 buffer at 4 ℃ for three hours. Subsequently, the protein complex was washed three times, eluted with 30 µl of 2 × SDS loading buffer and analyzed with Western Blotting assay.

Xu L., Wang J., Zhang D., Song L., Wu H., Wang J., Miao J., Guo H., Fang S., Si L., Chen J., Wu Y., Wu Y., Wang L., Zhang N., Chard L., Wang Y, & Cheng Z. (2022). The two-faced role of ATF2 on cisplatin response in gastric cancer depends on p53 context. Cell & Bioscience, 12, 77.

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In Vitro Protein Interaction Assay

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In vitro transcription and translation of goal, proteins were performed by using the TNT-coupled transcription and translation system (Promega Biotech Co., Ltd). Using a T7-TNT kit, we translated 1μg of pcDNA3.1 vector in the presents of methionine or 35 S-methionine in a reaction volume of 50μl. An aliquot of 10~20μl was used for each GST pull-down assay. Translation protein size was verified by subjecting 1~5μl reaction mixture to SDS-PAGE and western blot or autoradiography. The GST-pull down assay was performed by incubating equal amounts of GST, GST-fusion proteins immobilized by GST sepharose beads (Amersham Biosciences) with in vitro translated protein. Bound proteins were isolated by incubating the mixture for 2 h at 4℃, washing 3 times with binding buffer (20mM Tris, pH7.5, 50mM NaCl, 10% Glycerol, 1% NP-40). Proteins were eluted by 2×SDS loading buffer and separated by SDS-PAGE. Ponceau or Coomassie brilliant blue stain indicated the loading amounts of the GST-fusion proteins. The bound proteins were then visualized by western blot or autoradiography.

Pan X., Liu D., Ying M., Zheng G., Fan C., Pan F, & Ke Q. (2022). PAK5 is a potential target in myelodysplastic syndrome through interacting with LMO2 and GATA1. Cellular and molecular biology (Noisy-le-Grand, France), 68(9).

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CED-3 Protease Cleavage Assay

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CED-3 protease assays were done as described previously^{40 (link)}. Briefly, proteins of interest were synthesized and labeled with ³⁵S-Methionine using TNT Transcription and Translation coupled system (Promega) and incubated with or without 5 ng of purified CED-3 at 30°C for 1 hour. The reactions were then resolved by 15% SDS-PAGE gel and subjected to autoradiography.

Nakagawa A., Sullivan K.D, & Xue D. (2014). Caspase-activated phosphoinositide binding by CNT-1 promotes apoptosis by inhibiting the AKT pathway. Nature structural & molecular biology, 21(12), 1082-1090.

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CED-3 Protease Cleavage Assay

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