The detailed protocols were described elsewhere 33 (link). In brief, prior to import into purified mitochondria, [35S]-methionine and cysteine labeled Su9-DHFR (dihydrofolate reductase) fusion protein (based on the described protocol 33 (link), 52 (link), 53 (link), provided by Shanghai Genechem Co.) was generated with the TNT Quick Coupled Transcription/Translation kit (Promega, Shanghai, China). The isolated mitochondria (200 µg/mL) of human glioma cells was dissolved to the described import buffer 33 (link) and import reaction was started by the addition of translation mix. After 15 min, the import was terminated by trypsin on ice and soybean trypsin inhibitor was thereafter added. Mitochondria were pelleted by centrifugation, disrupted in Laemmli sample buffer and analyzed by SDS-PAGE and autoradiography.
Tnt quick coupled transcription translation kit
Manufactured by Promega
Sourced in United States
The TNT Quick Coupled Transcription/Translation kit is a laboratory tool that enables the concurrent synthesis of both mRNA and its corresponding protein from a DNA template in a single reaction vessel. The kit provides the necessary components, including RNA polymerase, amino acids, and energy sources, to facilitate this coupled transcription and translation process.
Automatically generated - may contain errors
Lab products found in correlation
Transcend non radioactive translation detection system, by Promega (3 mentions)
Protein purification kit, by Takara Bio (2 mentions)
Recombinant flag gpnmb, by OriGene (2 mentions)
Gst egfr, by Active Motif (2 mentions)
Recombinant active caspase 1, by R&D Systems (2 mentions)
Bl21 codonplus de3 ripl, by Agilent Technologies (2 mentions)
Hif 1α, by Novus Biologicals (2 mentions)
Lrrk2, by Sino Biological (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Phosphor screen, by GE Healthcare (1 mentions)
Fla 5100 scanner, by Fujifilm (1 mentions)
Gsh beads, by GE Healthcare (1 mentions)
Complete protease inhibitor cocktail, by Merck Group (1 mentions)
Glutathion sepharose beads, by GE Healthcare (1 mentions)
Bas 5000, by Fujifilm (1 mentions)
Genetools software, by Syngene (1 mentions)
Bio imaging system, by Syngene (1 mentions)
Amersham hyperfilm mp, by GE Healthcare (1 mentions)
Ek0031, by Thermo Fisher Scientific (1 mentions)
Anti myc antibody, by Thermo Fisher Scientific (1 mentions)
30 protocols using tnt quick coupled transcription translation kit
1
Mitochondrial Protein Import Assay
2
EGFR Binding to GST-IRF3 Complex
3
Recombinant Protein Expression and Purification
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Recombinant proteins were expressed in E.coli strain BL21-CodonPlus® (DE3)-RIPL (Agilent Technologies) and purified using Protein Purification Kit (Clontech). Recombinant Flag-GPNMB and PHD1 were purchased from Origene. GST-EGFR was purchased from Active Motif, HIF1α and BRK were purchased from Novus Biologicals. LRRK2 was purchased from SignalChem. Recombinant active Caspase-1 was purchased from R&D Systems. In vitro translation of LINK-A was conducted using TnT® Quick Coupled Transcription/Translation Kit and detection was performed using Transcend™ Non-Radioactive Translation Detection System (Promega).
4
In Vitro Protein Generation
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Human NRF2, MAFF, MAFG and MAFK proteins were generated by coupled in vitro transcription/translation system using their respective pcDNA3-based cDNA expression constructs and TNT Quick Coupled Transcription/Translation kit as recommended by the supplier (Promega, Madison, WI, USA).
5
Radiolabeled Protein Synthesis Protocol
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Prior to import into purified mitochondria, [35S]-methionine labeled precursor protein were generated with TNT Quick Coupled Transcription/Translation kit (Promega) and TRAN35S-LABEL, Metabolic Labeling Reagent (MP Biomedicals).
6
GST Pull-down Assay for Cnot3 Interactions
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To perform the GST pull down with GST-Aurora B and GST-ERK1 with Cnot3 and the deletion mutants of Cnot3, 0.5 μg of pCDNA-Cnot3, pCDNA-Cnot3 Δ1-200, pCDNA-Cnot3 Δ651-700, pCDNA-Cnot3 Δ701-751, and pCDNA-Cnot3 Δ651-751 were in vitro transcribed/translated using a TNT Quick Coupled Transcription/Translation kit (Promega, USA) according to the manufacturer’s instructions using 10 μCi of 35S-methionine to radiolabel the proteins. GST or GST-Aurora B or GST-ERK (1 μg) was added to the GSH beads (GE Healthcare, USA) in binding buffer (50 mM Tris-Cl, pH 8.0, 150 mM monopotassium glutamate, 1 mM EDTA, 0.1% Igepal CAL630, 5% glycerol, 0.2% BSA), supplemented with Complete protease inhibitor cocktail (Merck, USA), and incubated for 2 h at 4°C. The beads were then washed and 5 μl of the in vitro transcribed/translated CNOT3 was incubated with the beads overnight at 4°C. The beads were then washed with the binding buffer, and the proteins were eluted by boiling in loading buffer. The eluted proteins were subjected to SDS–PAGE, stained, dried for 1 h at 80°C, and exposed overnight to Phosphor screen in a cassette (GE Healthcare, USA). Images were captured in a Fujifilm FLA 5100 scanner (Japan) using Fujifilm FLA-5000 software.
7
Recombinant Protein Expression and Purification
8
Purification and Pull-Down Assay of Daxx Variants
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Combined His6+GST-tagged Daxx and its variants were produced in E. coli BL21 (DE3) from pET42b-based expression plasmids and purified with TALON (Clontech) and Glutathion-Sepharose beads (GE Healthcare). For the pull-down assays, these beads were incubated with in vitro translated 35S-labelled Brg1, prepared by TNT Quick Coupled Transcription/Translation kit (Promega) using TRAN 35S Label ( MP Radiochemicals). The bound proteins were eluted with the sample buffer, subjected to SDS-electrophoresis and visualised on a Phosphor Imaging Plate in BAS 5000 instrument (FujiFilm).
9
EMSA Analysis of PPAR-RXR Binding
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EMSA was performed using DIG Gel Shift Kit, 2nd (Roche) according to the manufacturer’s protocol. An oligonucleotide containing the rat ACO-PPRE and a random oligonucleotide were used as specific and non-specific controls, respectively. Synthesized wild-type and mutated oligonucleotides used for EMSA are listed in Table 1 . The mouse PPARα and RXRα proteins were generated from the expression vectors by in vitro transcription/translation using TNT® Quick Coupled Transcription/Translation Kit (Promega) according to the manufacturer’s protocol. The DNA-protein complexes were detected by chemiluminescence using Anti-Digoxigenin-AP Conjugate and CSPD (Roche) and quantified using a Bio-Imaging system (Syngene, Cambridge, UK) and Syngene GeneTools software.
10
FoxH1 Binding Site Validation
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EMSA tests were performed as described [31 (link)] using in vitro synthesized (TNT Quick-coupled Transcription/Translation kit, Promega). The following oligonucleotides containing putative FoxH1 binding sites were used: S1: CTGAATACACAAG ; S2: CCTTGTGTATTTGC ; S3: TGGTGTGTATTTA ; S4: TTTAATACACAACA . Oligos were end-labeled using γ-32P-Adenosine 5’-triphosphate (PerkinElmer) and polynucleotide kinase (Fermentas, EK0031). The binding reaction was performed using 25,000 cpm for each oligonucleotide, 2 μl protein in the presence of 0.1 mg/ml of poly(dI-dC) and 1 × binding buffer (5 × buffer: 60 mM HEPES pH 7.9, 20 mM Tris—HCl pH 7.9, 300 mM KCl, 60% (v/v) glycerol, 5 mM DTT and 5 mM EDTA). The mix was incubated for 45 min at room temperature. For competition assays, FoxH1 protein was 15 min pre-incubated with 100 fold higher concentration of unlabeled competitor oligos before adding the labeled probes. Binding was analyzed by running a native 5% polyacrylamide gel, followed by overnight exposure of film (GE Healthcare, Amersham Hyperfilm™ MP) at −80°C before development.
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