Dynorphin A1-8 (abbreviated dyn) and Leu-Enkephalin (LE) were purchased from Bachem (4005845 and 4006097, Torrance, CA); Met-Enkephalin (ME) was purchased from Sigma Aldrich (M6638, St. Louis, MO). Isotopically labeled leucine (13C615N1-leucine) was used to create an isotopically labeled dynorphin A 1-8internal standard (DYN*) through the University of Michigan’s protein synthesis core. Water, methanol, and acetonitrile for mobile phases are Burdick and Jackson HPLC grade purchased from VWR (Radnor, PA). All other chemicals were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise noted. Artificial cerebrospinal fluid (aCSF) consisted of 145 mM NaCl, 2.67 mM KCl, 1.4 mM CaCl2, 1.01 mM MgSO4, 1.55 mM Na2HPO4, and 0.45 mM Na2H2PO4 adjusted to pH 7.4 with NaOH. Ringer solution consisted of 148 mM NaCl, 2.7 mM KCl, 2.4 mM CaCl2, and 0.85 mM MgCl2 adjusted pH to 7.4 with NaOH. In experiments that used high K+ ringer solution NaCl was adjusted to 48 mM and KCl was adjusted to 100 mM, all other chemicals remained the same.
Met enkephalin
Manufactured by Merck Group
Sourced in Singapore, United States, Germany
Met-enkephalin is a peptide compound used in laboratory research. It functions as an opioid receptor agonist, interacting with specific receptors in the body to produce physiological effects. This compound is utilized by researchers to study the mechanisms and effects of opioid receptor activation in various experimental models.
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Lab products found in correlation
Forskolin, by Merck Group (2 mentions)
Pcdna4 to plasmid, by Thermo Fisher Scientific (1 mentions)
Naltrindole, by Bio-Techne (1 mentions)
Ab9026, by Abcam (1 mentions)
Snc80, by Bio-Techne (1 mentions)
Snc80, by Merck Group (1 mentions)
Pd98059, by Promega (1 mentions)
Goat anti mouse irdye 800, by Rockland Immunochemicals (1 mentions)
Fl 261, by Santa Cruz Biotechnology (1 mentions)
Pd98059, by Merck Group (1 mentions)
Morphine, by Merck Group (1 mentions)
Endomorphin 1, by Bio-Techne (1 mentions)
Tnf α, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Angiotensin 2, by Merck Group (1 mentions)
Val tyr val, by Merck Group (1 mentions)
Substance p, by Merck Group (1 mentions)
Leu enkephalin, by Merck Group (1 mentions)
5 protocols using met enkephalin
1
Quantification of Neuropeptide Dynamics
2
GPCR Signaling Pathway Plasmid Construction
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CXCL12 was purchased from Cedarlane. Forskolin, isobutylmethyl xanthine (IBMX), AVP, and met-enkephalin were purchased from Sigma. The following plasmids were already described: HA-CXCR447 (link), β-arrestin-2-LucII48 (link), β-arrestin-2-YFP49 (link), Gαi1−91RLucII47 (link), Gαs−117RLucII50 (link), GαoA−91RLucII51 (link), GFP10-Gγ152 (link), GFP10-Gγ253 (link), GFP10-EPAC-RLucII54 (link) and rGFP-CAAX33 (link). The cloning of CXCR4-RLuc and CXCR4-YFP in pcDNA3.1 was previously described11 (link). In the present study, CXCR4-RLuc and CXCR4-YFP were amplified and modified by PCR at the N-terminal end to add a myc epitope (EQKLISEEDL) or a HA epitope (YPYDVPDYA), respectively. Myc-CXCR4-RLuc and HA-CXCR4-YFP segments were then subcloned into pIREShyg3 (BsrG1/AflII) and pIRESpuro3 (Nhe1/AflII) respectively. The human μOR and V2R were amplified with a SNAP tag at their N-terminal (NEB) and subcloned in the pcDNA4/TO plasmid (Invitrogen). All the mutants were obtained by site-directed mutagenesis using the extension of overlapping gene segments by PCR technique and validated by sequencing.
3
Antibody Characterization in Cellular Studies
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Antibodies used in this study were as follows: rabbit polyclonal anti-GFP antibody (no. ab290, Abcam, Hong Kong); mouse monoclonal anti-KRT10, clone DE-K10 (Ivanyi et al., 1989 (link); no. ab9026, Abcam, or no. MS-611, Thermo Fisher Scientific, Fremont, CA); mouse monoclonal anti-KRT1 antibody, clone 34βB4 (no. NCL-CK1, Novocastra, New Castle Upon Tyne, UK); mouse monoclonal anti-IVL (Sy5, no. MS-126, Thermo Fisher Scientific), rabbit anti-proliferating cell nuclear antigen (FL-261, no. sc-7907, Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal anti-desmoplakin, clone 11-5F (kindly provided by D. Garrod, University of Manchester, UK (Parrish et al., 1987 (link))); and mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (3E8AD9, no. A21994, Life Technologies). Secondary antibodies used were goat anti-mouse AlexaFluor 594, goat anti-rabbit AlexaFluor 488, and goat anti-rabbit AlexaFluor 594 (Life Technologies/Molecular Probes, Eugene, OR). Detection antibodies used for western blotting were goat anti-mouse IRDye 800 and goat anti-rabbit IRDye 700DX (Rockland, Gilbertsville, PA). SNC80 and naltrindole were from Tocris Biosciences (Bristol, UK), PD98059 from Promega (Madison, WI), and the solvent for SNC80 and PD98059, DMSO, as well as Met-enkephalin from Sigma-Aldrich (Singapore).
4
Comparative Analysis of MOR Agonists
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We chose six MOR agonists that include endogenous and exogenous ligands and that vary in terms of G-protein bias: morphine (Sigma–Aldrich; St. Louis, MO, USA), DAMGO ([D-Ala2-MePhe4-Gly-ol]-enkephalin; Bachem, Torrance, CA, USA), met-enkephalin (Sigma–Aldrich), β-endorphin (American Peptide Co., Sunnyvale, CA, USA), endomorphin-1 (Tocris Bioscience, Minneapolis, MN, USA), and TRV130 (oliceridine; synthesized by B.E.B.). All ligands except TRV130, which was first dissolved in DMSO (20%), were dissolved in water, then prepared as serial dilutions to concentrations between 10−11 to 10−5 M. GPCR independent substrates including forskolin, TNF-α and PMA (Sigma–Aldrich) were used to activate cAMP, NF-ĸB and MAPK/JNK respectively. All GPCR independent substrates were diluted in DMSO to final concentrations of 10 μM for forskolin, 50 ng/mL for TNF-α, and 10 ng/mL for PMA.
5
Purity Evaluation of Lichen Metabolites
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Matrix reagent dithranol was purchased from Avocado Research Chemicals Ltd (Morecombe, UK). HCCA, DHB, Gramicidin A-D, substance P and the HPLC peptide mix (containing 0.5 mg of the following compounds: Angiotensin II, Gly-Tyr, Leu enkephalin, Met enkephalin and Val-Tyr-Val) were all purchased from Sigma-Aldrich (Steinheim, Germany). Small organic analytes were obtained from former phytochemical research projects or generously provided by collaborators. Likewise, lichen metabolites considered in the study were isolated during previous phytochemical investigations. Placodiolic acid and didymic acid were kindly provided by Dr M. Millot (Université de Limoges, Limoges, France). Citreorosein, porphyrilic acid and schizopeltic acid were generous gifts from Dr H. J. Sipman (Huneck's compounds library -National History Museum -Berlin, Germany).
Purity evaluation of the lichen metabolites was based on 1 H NMR experiments. Each compound exhibited a degree of purity of at least 95%.
Purity evaluation of the lichen metabolites was based on 1 H NMR experiments. Each compound exhibited a degree of purity of at least 95%.
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