The 18-mer TFOs (3′-GGAAGGNZN’GAGGAGGGA-5′) (NN′ = GA, GG, AG and AA) incorporating dAN derivatives (Z = aminoethyl-dAN, 3-phenol-dAN and 2-phenol-dAN) were synthesized on a 1 μmol scale by an automated DNA synthesizer (Nihon Techno Service Co., Ltd.) using standard phosphoramidite chemistry. Cleavage from the resin was accomplished by an overnight treatment with 28% ammonium hydroxide at 55 °C for 5 h, followed by reversed-phase HPLC purification (column: Nacalai Tesque COSMOSIL 5C18-ARII, 10 × 250 mm, solvents: A: 0.1 M TEAA buffer, B: CH3CN, gradient: B for 10% to 40%/20 min, flow rate: 3.0 mL min−1, UV: 254 nm, column oven: 35 °C). The DMTr group was deprotected in 5% aqueous acetic acid at room temperature for 30 min, followed by reversed-phase HPLC purification (HPLC conditions: column: Nacalai Tesque COSMOSIL 5C18-ARII, 4.6 × 250 mm, solvents: A: 0.1 M TEAA buffer, B: CH3CN, B: 5% to 30%/20 min, flow rate: 1.0 mL min−1, UV: 254 nm, column oven: 35 °C). The structural integrity of the synthesized oligonucleotides was analyzed by MALDI-TOF MS (Table S1 and see an ESI† ).
Cosmosil 5c18 ar 2
Manufactured by Nacalai Tesque
Sourced in Japan
Cosmosil 5C18-AR-II is a reversed-phase high-performance liquid chromatography (HPLC) column. It features a C18 bonded silica stationary phase and is designed for analytical separations.
Automatically generated - may contain errors
Lab products found in correlation
Silica gel 60n, by Kanto Chemical (3 mentions)
Waters 600, by Waters Corporation (2 mentions)
Cosmonice filter, by Nacalai Tesque (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Sodium 1 13c acetate, by Cambridge Isotopes (1 mentions)
1 13c acetate, by Cambridge Isotopes (1 mentions)
1 4 13c2 succinic acid, by Cambridge Isotopes (1 mentions)
Cosmosil 75c18 prep, by Nacalai Tesque (1 mentions)
Ft ir 460 plus spectrometer, by Jasco (1 mentions)
Cosmosil 75c18 opn, by Nacalai Tesque (1 mentions)
Dowex 50wx2 100, by Merck Group (1 mentions)
Sephadex lh 20, by Merck Group (1 mentions)
Rp 18f254 plates, by Merck Group (1 mentions)
Avance 500 spectrometer, by Bruker (1 mentions)
Prominence high performance liquid chromatography, by Shimadzu (1 mentions)
Methanol, by Merck Group (1 mentions)
Ecz600 spectrometers, by JEOL (1 mentions)
Pack sil 06, by YMC (1 mentions)
Diaion hp 20, by Mitsubishi (1 mentions)
Sephadex lh 20, by GE Healthcare (1 mentions)
31 protocols using cosmosil 5c18 ar 2
1
Synthesis and Purification of 18-mer TFOs
2
Synthesis and Characterization of Dipeptides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four dipeptides, Leu-Hyp, Pro-Hyp, Glu-Hyp and Phe-Hyp, were synthesized by a liquid-phase method [15 ]. When synthesizing Leu-Hyp, 1.8 mM of Boc-Leucine-OH (Watanabe chemical industry, Hiroshima, Japan) was reacted with 1.8 mM of (t-Butyl)-trans-4-hydroxy-L-proline t-butyl ester hydrochloride (Watanabe chemical industry) overnight at 4℃ in the presence of 2.6 mM of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide monohydrochloride (Watanabe chemical industry) and 4.4 mM of 1-hydroxybenzotriazole (Watanabe chemical industry) as catalysts. The reaction product, N and C-protected dipeptidyl Leu-Hyp derivative (Boc-Leu-Hyp-OtBu), was reacted with 4 M HCl/dioxane (30 min, 25℃) to cleave Boc groups and OtBu groups [16 (link)]. The obtained crude product was purified by preparative reversed-phase HPLC using Cosmosil 5C18-AR-II (10 mm I.D. × 250 mm; Nacalai Tesque).The final product was characterized by analytical reversed-phase HPLC using Cosmosil 5C18-AR-II (2.0 mm I.D. × 250 mm; Nacalai Tesque) and electrospray ionization ion-trap mass spectrometry with HPLC linked to 3200 Q Trap LC-MS/MS system. Pro-Hyp, Glu-Hyp and Phe-Hyp were synthesized in the same manner as Leu-Hyp, using the starting materials corresponding to the dipeptide to be synthesized.
3
Solid-phase Oligonucleotide Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Solid-phase oligonucleotide synthesis was performed on an ABI DNA synthesizer (Applied Biosystem, Foster City, CA) or M-2-MX DNA/RNA synthesizer (Nihon Techno Service Co., Ltd, Tsukuba, Japan). 1C and IM-CT were synthesized on solid support using FPdC phosphoramidite (compound 10 ) and commercially available O5′-dimethoxytrityl-2′-deoxyribonucleoside O3′-phosphoramidites. Cleavage from the solid support and deprotection, followed by purification with Glen-PakTM cartridges yielded the desired oligonucleotides. After HPLC purification (COSMOSIL 5C18 AR-II, Nacalai Tesque, Inc., Kyoto, 150 × 10 mm id), the identity of the products were confirmed by MALDI-TOF MS using a Bruker microflex-KSII (Bruker Corporation, Billerica, MA) (Table S2, ESI† ). DNA concentrations were determined by using NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE).
4
Isotope Labeling and NMR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sodium [1-13C]acetate, [1-13C]acetate, sodium [1-13C]propionate, and [1,4-13C2]succinic acid were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). 13C NMR spectra were obtained on a Bruker AVANCE 400 spectrometer (Bruker BioSpin K.K., Yokohama, Japan). Cosmosil 75C18-PREP (Nakalai Tesque, Inc., Kyoto, Japan, 70 μm) was used for ODS column chromatography. HPLC separation was performed using a Cosmosil 5C18-AR-II (Nacalai Tesque Inc., 20 × 250 mm, Kyoto, Japan) with a photodiode array detector.
5
Heme Biotinylation Protocol
6
Spectroscopic Analysis of Organic Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Melting points were determined on a Yanaco Micro MP apparatus. Optical rotations were measured on a JASCO P-2100 polarimeter (Hachioji, Tokyo, Japan). Infrared spectra were recorded on JASCO FT/IR-460 Plus spectrometer. 1D and 2D NMR were carried out using Bruker Avance 500 spectrometer (Bruker, Mass., Billerica, MA) with tetramethylsilane as an internal standard. ESI-MS data including high-resolution mass spectrum were measured on Shimadzu LCMS-IT-TOF spectrometer (Kyoto, Japan) HREIMS was recorded on a JEOL MStation JMS-700 spectrometer (JEOL Ltd., Tokyo, Japan). Column chromatography was performed using silica gel (Kanto, 40–50 μm, Tokyo, Japan), Cosmosil 75C18-OPN (Nacalai Tesque Inc., Kyoto, Japan) and Sephadex LH-20 (Dowex® 50WX2-100, Sigma-Aldrich, Tokyo, Japan). Analytical TLC was performed on pre-coated silica gel 60F254 and RP-18 F254 plates (0.25 or 0.50 mm thickness, Merck KGaA, Darmstadt, Germany). Cosmosil 5C18-AR-II (Nacalai Tesque Inc., Kyoto, Japan) was used for analytical and semi-preparative HPLC (250 × 4.6 mm for analytical HPLC, and 250 × 10.0 mm for semi-preparative HPLC).
7
Recombinant Proinsulin Purification from E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant human proinsulin was expressed as inclusion bodies in Escherichia coli cells. Purification of proinsulin was carried out as previously described (Okumura et al., 2011 (link)). Briefly, inclusion bodies were treated with 100 mM Tris/HCl buffer, pH 8.0, containing 8 M urea and 10 mM DTT, and the solution was stood for 3 hr at 50°C. Reduced and denatured proinsulin was purified by RP-HPLC using Cosmosil 5C18-AR-II (4.6 mm I.D. ×250 mm, Nacalai Tesque) monitored at 220 nm. Molecular mass of purified proinsulin was calculated using ProteinProspectors (http://prospector.ucsf.edu/prospector/mshome.htm ) and its identity was confirmed by MALDI-TOF/MS. Purified proinsulin was lyophilized at −80°C until used.
8
Purification and Characterization of Organic Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemical regents and
solvents were purchased
from commercial sources and used without further purifications. The 1H NMR spectra were recorded on a JEOL ECX-500II spectrometer
(Tokyo, Japan) with TMS as an internal standard. Purification was
performed using a Prominence high-performance liquid chromatography
(HPLC) system (Shimadzu Corp., Kyoto, Japan) equipped with Cosmosil
5C18-AR-II (4.6 × 150 mm, Nacalai Tesque, Kyoto, Japan) or Shodex
Asahipak GF-310HQ (7.5 × 300 mm, Showa Denko K. K., Tokyo, Japan)
columns. As the mobile phase, acetonitrile/H2O containing
0.1% trifluoroacetic acid (AR-II) and acetonitrile (GF-310) were used
at a flow rate of 1.0 and 0.5 mL/min, respectively.
solvents were purchased
from commercial sources and used without further purifications. The 1H NMR spectra were recorded on a JEOL ECX-500II spectrometer
(Tokyo, Japan) with TMS as an internal standard. Purification was
performed using a Prominence high-performance liquid chromatography
(HPLC) system (Shimadzu Corp., Kyoto, Japan) equipped with Cosmosil
5C18-AR-II (4.6 × 150 mm, Nacalai Tesque, Kyoto, Japan) or Shodex
Asahipak GF-310HQ (7.5 × 300 mm, Showa Denko K. K., Tokyo, Japan)
columns. As the mobile phase, acetonitrile/H2O containing
0.1% trifluoroacetic acid (AR-II) and acetonitrile (GF-310) were used
at a flow rate of 1.0 and 0.5 mL/min, respectively.
9
Quantification of Capsaicin via HPLC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Capsaicin was quantified using reverse-phase HPLC (Waters 600S, Waters, Milford, MA, USA) performed in a C18 column (150 mm × 4.6 i.d., Cosmosil 5C18-AR-II, Nacalai Tesque Inc., Kyoto, Japan). A stock capsaicin solution was serially diluted with methanol from 0 to 50 µg/mL (Sigma-Aldrich, St. Louis, MO, USA) to prepare a calibration curve (R2 ≥ 0.99). The analytical conditions were as follows: mobile phase, methanol/distilled water (80:20), isocratic; flow rate, 1.0 mL/min; injection volume, 10 μL; and detection wavelength, 222 nm.
10
Purification of Natural Products
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NMR spectra were recorded on JEOL ECZ600 spectrometers in a deuterated solvent whose chemical shift was used as an internal standard. Column chromatography was performed using Silica Gel 60N (Kanto Chemical Co., Tokyo, Japan), Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai, Japan), Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan) and Sephadex LH-20 (GE Healthcare Japan, Tokyo, Japan). Preparative HPLC was performed using YMC-Pack SIL-06 (YMC Co. Ltd., Kyoto, Japan), COSMOSIL 5C18-AR-II, COSMOSIL Cholester, COSMOSIL πNAP (Nacalai Tesque, Inc. Kyoto, Japan) and CAPCELL PAK C18 MGII (OSAKA SODA Ltd., Osaka, Japan).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!