Linearized pGEMT-MT1DP was used as the template for MT1DP transcription, and MT1DP RNAs were then produced through in vitro transcription using Ambion mMESSAGE mMACHINE T7 Transcription Kit following the manufacturer’s instructions (Invitrogen). MT1DP RNAs were labeled with biotin, and proteins were pulled down from cell lysates using a RNA-Protein Pull-Down kit on the basis of manufacturer’s instructions (Thermo Fisher Scientific, USA). RNA-pulled down proteins were separated by SDS-PAGE, and then differentially presented bands were analyzed by MS at Beijing Protein Institute.
Ambion mmessage mmachine t7 transcription kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ambion mMESSAGE mMACHINE T7 Transcription Kit is a laboratory product designed for in vitro transcription of RNA from DNA templates. The kit provides the necessary components, including the T7 RNA polymerase enzyme, to efficiently generate high-quality and capped RNA transcripts.
Automatically generated - may contain errors
Lab products found in correlation
Geneart, by Thermo Fisher Scientific (2 mentions)
Rna protein pull down kit, by Thermo Fisher Scientific (1 mentions)
Ranolazine dihydrochloride, by Merck Group (1 mentions)
Q5 site directed mutagenesis kit, by Thermo Fisher Scientific (1 mentions)
Flecainide acetate salt, by Merck Group (1 mentions)
Mexiletine hydrochloride, by Merck Group (1 mentions)
Nanoliter injector, by World Precision Instruments (1 mentions)
Tricaine methanesulfonate ms 222, by Merck Group (1 mentions)
Collagenase type a, by Roche (1 mentions)
10 protocols using ambion mmessage mmachine t7 transcription kit
1
Quantitative Proteomics of MT1DP Interactome
2
Heterologous Expression of nAChR Subunits in Xenopus Oocytes
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The plasmids containing different kinds of nAChR subunits were transferred into DH5α competent cells, then they were extracted and digested with the appropriate restriction enzymes. The linearized DNA templates were purified, and their concentration and purity were determined by the gel electrophoresis method. The Invitrogen™ Ambion™ mMESSAGE mMACHINE™ T7 Transcription Kit was used for transcription, and the transcribed RNA was purified to obtain the final cRNA. RNA concentration and purity were assessed using the gel electrophoresis method. The pre-prepared RNAs of different subunits were mixed and injected into Xenopus laevis oocytes as described previously [40 (link)]. After injection, oocytes were cultured in ND96 solution (96 mmol/L NaCl, 2 mmol/L KCl, 1.8 mmol/L CaCl2, 1 mmol/L MgCl2, 5 mmol/L HEPES, pH 7.1–7.7) containing 1% penicillin (10 μg/mL), 1% streptomycin (10 μg/mL), 1% gentamicin (100 μg/mL) at 17 °C for 2–5 days, then the currents were detected by the TEVC. In this experiment, the animal assay was in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Hainan University.
3
Rescue of s741 Phenotypes Using Furinb mRNA
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To rescue s741 phenotypes using WT furinb mRNA, a full‐length furinb complementary DNA (cDNA) was amplified by PCR from WT total cDNA and inserted into the RNA synthesis plasmid pCS2+. Sense RNA was synthesized using the Ambion mMessage mMachine T7 transcription kit (Invitrogen). A total of 20–50 pg of capped furinb RNA was injected into the yolk of embryos from s741 heterozygotes incrosses at the 1–4 cell stage. The injected larvae were fixed in 4% paraformaldehyde at 96 hpf and genotyped by PCR.
The Tg(Tp1:wt‐furinb‐EGFP), Tg(Tp1:s741‐furinb‐EGFP), Tg(fabp10a:wt‐furinb‐GFP), and Tg(fabp10a:s741‐furinb‐EGFP) transgenic constructs were generated using the multisite gateway‐based Tol2kit.[19 ]
The Tg(Tp1:wt‐furinb‐EGFP), Tg(Tp1:s741‐furinb‐EGFP), Tg(fabp10a:wt‐furinb‐GFP), and Tg(fabp10a:s741‐furinb‐EGFP) transgenic constructs were generated using the multisite gateway‐based Tol2kit.[
4
Rescue of s741 phenotypes using furinb mRNA
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted February 26, 2022. ; https://doi.org/10.1101/2022.02.24.481764 doi: bioRxiv preprint
To rescue s741 phenotypes using WT furinb mRNA, a full-length furinb cDNA was amplified by PCR from WT total cDNA and inserted into the RNA synthesis plasmid pCS2+. Sense RNA was synthesized from the XhoI-linearized plasmid using the Ambion mMessage mMachine T7 transcription kit (Invitrogen). 20-50 pg of capped furinb RNA was injected into the yolk of embryos from s741 heterozygotes incrosses at the 1-4 cell stage. The injected larvae were fixed in 4% PFA at 96 hpf and genotyped by PCR.
The Tg(Tp1:wt-furinb-EGFP) and Tg(Tp1:s741-furinb-EGFP) transgenic constructs were generated using the multisite gateway-based Tol2kit (Kwan et al., 2007) . The constructs were individually injected into the 10 WT Tg(Tp1:ras-mCherry) embryos at the one-cell stage and imaged live using confocal at 120 hpf.
The copyright holder for this preprint this version posted February 26, 2022. ; https://doi.org/10.1101/2022.02.24.481764 doi: bioRxiv preprint
To rescue s741 phenotypes using WT furinb mRNA, a full-length furinb cDNA was amplified by PCR from WT total cDNA and inserted into the RNA synthesis plasmid pCS2+. Sense RNA was synthesized from the XhoI-linearized plasmid using the Ambion mMessage mMachine T7 transcription kit (Invitrogen). 20-50 pg of capped furinb RNA was injected into the yolk of embryos from s741 heterozygotes incrosses at the 1-4 cell stage. The injected larvae were fixed in 4% PFA at 96 hpf and genotyped by PCR.
The Tg(Tp1:wt-furinb-EGFP) and Tg(Tp1:s741-furinb-EGFP) transgenic constructs were generated using the multisite gateway-based Tol2kit (Kwan et al., 2007) . The constructs were individually injected into the 10 WT Tg(Tp1:ras-mCherry) embryos at the one-cell stage and imaged live using confocal at 120 hpf.
5
Plasmid DNA Preparation and cRNA Synthesis
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Plasmid DNAs were purchased from GeneArt (Thermo Fisher scientific), General biosystems Inc. or Twist Bioscience. All gene constructs were sub-cloned into either the pUNIV or pcDNA3.1+ backbone. pUNIV backbone was a gift from Cynthia Czajkowski (Addgene plasmid # 24705; http://n2t.net/addgene:24705 ; RRID:Addgene_24705). Conventional site-directed mutagenesis was performed using standard PCR. Primer sequences are listed in Supplementary Table 2 . Complementary RNA (cRNA) for oocyte microinjection was transcribed from respective linearized cDNA using the Ambion mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific).
6
Generation and Characterization of Gene Constructs
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Gene constructs were generated and used as described in ref. 23 (link); see supplementary information for sequence details. Standard site-directed mutagenesis was performed using PCR, and deletions were created using the Q5 site-directed mutagenesis kit (Thermo Fisher Scientific). The complementary DNA was linearized and transcribed into complementary RNA (cRNA) for oocyte microinjection using the Ambion mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific).
All chemicals were purchased from Sigma-Aldrich, Iris Biotech, Rapp polymere, Combi-Blocks and Chem-Impex unless specified otherwise. AADs were purchased from Sigma-Aldrich (quinidine sulfate salt dihydrate, catalog number: Q0875; mexiletine hydrochloride, catalog number: M2727; flecainide acetate salt, catalog number: F6777; ranolazine dihydrochloride, catalog number R6152) and stored according to product specification. Substances were dissolved in ND96 solution (see Two-Electrode Voltage Clamp Recordings section below for composition) and diluted to the specified concentrations, and the pH value was adjusted to 7.4. The prepared solutions were then stored at room temperature and used for no longer than 20 d.
All chemicals were purchased from Sigma-Aldrich, Iris Biotech, Rapp polymere, Combi-Blocks and Chem-Impex unless specified otherwise. AADs were purchased from Sigma-Aldrich (quinidine sulfate salt dihydrate, catalog number: Q0875; mexiletine hydrochloride, catalog number: M2727; flecainide acetate salt, catalog number: F6777; ranolazine dihydrochloride, catalog number R6152) and stored according to product specification. Substances were dissolved in ND96 solution (see Two-Electrode Voltage Clamp Recordings section below for composition) and diluted to the specified concentrations, and the pH value was adjusted to 7.4. The prepared solutions were then stored at room temperature and used for no longer than 20 d.
7
Heterologous Expression of Neurotransmitter Transporters
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The DNA encoding the mouse ATB (gift of V. Ganapathy, Texas Tech University Health Sciences Center, Lubbock, TX), the rat GlyT2a (gift of B. Lopez Corcuera and C. Aragon, Universidad Autónoma de Madrid, Madrid, Spain), and the rat GlyT1b (gift of Kelli E. Smith, Synaptic Pharmaceutical Corp., Paramus, NJ) were subcloned in a modified pRC/CMV vector as described previously in ref. 15 (link). Capped cRNAs were synthesized using the Ambion mMessage mMachine T7 transcription kit (Thermo Fisher Scientific) and kept at 1 µg/µL at –80 ∘C.
Female Xenopus laevis were anesthetized using 0.1% Tricaine methanesulfonate (MS222, Sigma) dissolved in 0.1% NaHCO3 and maintained on ice. Defolliculated oocytes were harvested from ovaries by shaking incubation in Ca -free OR-2 solution (in mM: 85 NaCl, 1 MgCl2, 5 Hepes, pH 7.6, with KOH) containing 5 mg mL– 1 collagenase (type A, Roche). Oocytes were kept at 19 ∘C in a Barth’s solution (in mM: 88 NaCl, 1 KCl, 0.41 CaCl2, 0.82 MgSO4, 2.5 NaHCO3, 0.33 CaNO3, 5 Hepes, pH 7.4 [with NaOH]) containing 50 µg/mL gentamycin. cRNAs (50 ng) were injected using a nanoliter injector (World Precision Instruments).
Female Xenopus laevis were anesthetized using 0.1% Tricaine methanesulfonate (MS222, Sigma) dissolved in 0.1% NaHCO3 and maintained on ice. Defolliculated oocytes were harvested from ovaries by shaking incubation in Ca -free OR-2 solution (in mM: 85 NaCl, 1 MgCl2, 5 Hepes, pH 7.6, with KOH) containing 5 mg mL– 1 collagenase (type A, Roche). Oocytes were kept at 19 ∘C in a Barth’s solution (in mM: 88 NaCl, 1 KCl, 0.41 CaCl2, 0.82 MgSO4, 2.5 NaHCO3, 0.33 CaNO3, 5 Hepes, pH 7.4 [with NaOH]) containing 50 µg/mL gentamycin. cRNAs (50 ng) were injected using a nanoliter injector (World Precision Instruments).
8
Xenopus Oocyte Expression Vector Construction
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A Xenopus oocyte expression vector, pNCB1 (GenBank accession number MF984401), was constructed based on pGEMHE [32 (link)], which contains a T7 promoter, β-globin 5′-UTR, multiple cloning sites, β-globin 3′-UTR, and a poly-A sequence. Native CM2 [Locus, YP_089658.1 (C/Ann Arbor/1/50)] and native and modified DM2 [Locus, AFJ19025 ((D/Swine/Oklahoma/1334/2011)] were synthesized by adding sequences corresponding to a restriction enzyme site immediately before and after the coding sequence and were cloned into pNCB1. BamHI and XbaI were used for 5′ and 3′ ends, except in the case of CM2 (SmaI was used at the 5′ end). DNA was prepared by GenScript (Piscataway, NJ, USA). Before mRNA preparation, pNCB plasmid clones were linearized by HindIII and recovered by ethanol precipitation using half volume of 5 M ammonium acetate and two volumes of 100% ethanol. Invitrogen Ambion mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher, Waltham, MA, USA) was used for mRNA synthesis. mRNA was recovered by LiCl precipitation and dissolved into PBS comprising 137 mM NaCl, 2.7 mM KCl, and 1.8 mM Na2HPO4. mRNA was stored in −20°C.
9
Plasmid Construct Preparation for Oocyte Microinjection
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Plasmid DNAs were purchased from GeneArt (ThermoFisher scientific), General biosystems Inc. or Twist Bioscience. All gene constructs were sub-cloned into either the pUNIV or pcDNA3.1+ backbone. pUNIV backbone was a gift from Cynthia Czajkowski (Addgene plasmid # 24705; http://n2t.net/addgene:24705; RRID:Addgene_24705).
Conventional site-directed mutagenesis was performed using standard PCR.
Complementary RNA (cRNA) for oocyte microinjection was transcribed from respective linearized cDNA using the Ambion mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific).
Conventional site-directed mutagenesis was performed using standard PCR.
Complementary RNA (cRNA) for oocyte microinjection was transcribed from respective linearized cDNA using the Ambion mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific).
10
Xenopus Oocyte Protein Expression
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Protein expression in Xenopus oocytes was carried out as previously described [83, 84] . In brief, cDNA coding for human CAIX, rat MCT1, rat MCT2, rat MCT4, rat CD147-WT or a mutant of the protein, as well as rat GP70-WT or mutant, all cloned into the Xenopus expression vector pGEM-He-Juel, was transcribed in vitro using the Invitrogen™ Ambion™ mMESSAGE mMACHINE™ T7 Transcription Kit (Thermo Fisher). Xenopus laevis females were purchased from the Radboud University, Nijmegen, Netherlands. The procedure for surgical removal of oocytes from anaesthetized frogs was approved by the Landesuntersuchungsamt Rheinland-Pfalz, Koblenz (23 177-07/A07-2-003 §6) and the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg (33.19-42502-05-17A113). Oocytes were singularized by collagenase treatment for 1 h at 28°C in Ca 2+ -free oocyte saline. Singularized oocytes were stored in Ca 2+ -containing oocyte saline (82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl 2 , 1mM MgCl 2 , 1mM Na 2 HPO 4 , 5mM HEPES, pH 7.8) at 18°C. Oocytes of the developmental stages IV and V were injected with 5 ng of cRNA coding for MCT1, MCT2, or MCT4, together with 10 ng of cRNA coding for rCD147-WT or a mutant of rCD147, or rGP70-WT or a mutant of rGP70, and 5 ng of cRNA coding for CAIX-WT.
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