For whole RHA1 lipid content analysis, cells were harvested during the transition from exponential to stationary growth phases, washed with a 150 mM NaCl and suspended in 50 mM Tris-HCl, pH 7.0, and 300 mM NaCl buffer. Cells were subjected to four rounds of 60 s of bead beating using a FastPrep-24 bead beater (MP Biomedicals, Solon, OH) set to 5.5. The sample was incubated for 5 min on ice between rounds. To identify lipids, samples of whole cells or isolated LDs were extracted with chloroform-methanol (2:1, v/v). Aliquots were analyzed by thin layer chromatography (TLC) on 60F254 silica gel plates (Merck) using hexane/diethyl ether/acetic acid (80:20:1, v/v/v) as a solvent system. Triolein (Sigma-Aldrich, T7140) was used as the TAG standard. The plates were dried at room temperature for 10 min and immediately sprayed with a 10% cupric sulfate in 8% phosphoric acid solution. The plates were then incubated in an oven at 150 °C for 10 min.
60f254 silica gel plate
Manufactured by Merck Group
Sourced in Germany
The 60F254 silica gel plates are a type of thin-layer chromatography (TLC) plate used for analytical and preparative separations. These plates are coated with a layer of silica gel containing a fluorescent indicator, which allows for the visualization of separated compounds under UV light. The plates provide a stable and inert surface for the separation of a variety of chemical compounds.
Automatically generated - may contain errors
Lab products found in correlation
Triolein, by Merck Group (1 mentions)
Fastprep 24 bead beater, by MP Biomedicals (1 mentions)
Autochro 3000, by Younglin (1 mentions)
Prodigy ods 2 c18 column, by Phenomenex (1 mentions)
Q star xl hybrid ms ms system, by Thermo Fisher Scientific (1 mentions)
6130 single quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Agilent chemstation software, by Agilent Technologies (1 mentions)
1100 hplc system, by Agilent Technologies (1 mentions)
Isolera, by Biotage (1 mentions)
Photodiode array detector, by Agilent Technologies (1 mentions)
Fischer johns block, by Thermo Fisher Scientific (1 mentions)
Avance 300 mhz spectrometer, by Bruker (1 mentions)
Snap kp sil cartridge, by Biotage (1 mentions)
Gemini 300 mhz spectrometer, by Agilent Technologies (1 mentions)
Bx series ft ir spectrometer, by PerkinElmer (1 mentions)
Compass dataanalysis 4, by Bruker (1 mentions)
Avance spectrometer, by Bruker (1 mentions)
1600 ft ir spectrometer, by PerkinElmer (1 mentions)
Avance 3 spectrometer, by Bruker (1 mentions)
Hp1100, by Agilent Technologies (1 mentions)
29 protocols using 60f254 silica gel plate
1
Analysis of Whole-Cell RHA1 Lipid Content
2
Qualitative and Quantitative Ginsenoside Analysis
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TLC analysis. A reaction solution containing ginsenoside was extracted with an equal volume of water-saturated n-butanol; after centrifugation, the n-butanol fraction was examined by TLC using 60F254 silica gel plates (Merck, Germany) and CHCl3-CH3OH-H2O (65:35:10, v/v/v, lower phase) as the solvent. TLC plates were sprayed with 10% (v/v) H2SO4, followed by heating at 110°C for 5 min to visualize ginsenoside spots, which were identified by comparing with a standard.
HPLC analysis. HPLC analysis of ginsenosides was performed using AutoChro 3000 software (Younglin, Korea) equipped with a quaternary pump, automatic injector, and single-wavelength UV detector (model 730D) for peak identification and integration. The HPLC column used was a Prodigy ODS (2) C18 column (4.6 × 150 mm, 5 μm) (Phenomenex, USA) combined with an Agilent safeguard column. Isocratic elution was performed, using acetonitrile (A ) and water (B ) at a ratio of 34:66 (v/v) as mobile phase, for 20 min, at a flow rate of 1.0 ml/min. Detection was performed by monitoring absorbance at 203 nm.
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3
Analytical Characterization of α-Noscapine
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Reagents and
all solvents used
were analytically pure. Air-sensitive reagents were transferred by
a syringe or double-ended needle. Evaporation of solvents was performed
at reduced pressure by using a heidolph rotary evaporator. TLC (precoated
silica plates and visualizing under UV light) is used to monitor progress
of the reactions. 1H and 13C NMR spectra of
samples in CDCl3 were recorded on an AVANCE-300, 400, 500
MHz spectrometer. Chemical shifts presented are relative to an internal
standard TMS (δ = 0.0). Spin multiplicities are described as
s (singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet),
or m (multiplet). Coupling constants are reported in hertz (Hz). Mass
spectra were recorded in ESI conditions at 70 eV on an LC-MSD (Agilent
technologies) spectrometer. All high-resolution spectra were recorded
on the QSTAR XL hybrid MS/MS system (Applied Bio systems/MDS sciex,
Foster
city, USA), equipped with an ESI source (CSIR-IICT, Hyderabad). Column
chromatography was performed on silica gel (60–120 mesh) supplied
by Acme Chemical Co., India. TLC was performed on Merck 60 F-254 silica
gel plates. Commercially available anhydrous solvents dichloromethane,
methanol, acetone, and ethyl acetate were used as such. Natural α-noscapine
was procured from Sigma-Aldrich.
all solvents used
were analytically pure. Air-sensitive reagents were transferred by
a syringe or double-ended needle. Evaporation of solvents was performed
at reduced pressure by using a heidolph rotary evaporator. TLC (precoated
silica plates and visualizing under UV light) is used to monitor progress
of the reactions. 1H and 13C NMR spectra of
samples in CDCl3 were recorded on an AVANCE-300, 400, 500
MHz spectrometer. Chemical shifts presented are relative to an internal
standard TMS (δ = 0.0). Spin multiplicities are described as
s (singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet),
or m (multiplet). Coupling constants are reported in hertz (Hz). Mass
spectra were recorded in ESI conditions at 70 eV on an LC-MSD (Agilent
technologies) spectrometer. All high-resolution spectra were recorded
on the QSTAR XL hybrid MS/MS system (Applied Bio systems/MDS sciex,
Foster
city, USA), equipped with an ESI source (CSIR-IICT, Hyderabad). Column
chromatography was performed on silica gel (60–120 mesh) supplied
by Acme Chemical Co., India. TLC was performed on Merck 60 F-254 silica
gel plates. Commercially available anhydrous solvents dichloromethane,
methanol, acetone, and ethyl acetate were used as such. Natural α-noscapine
was procured from Sigma-Aldrich.
4
Analytical Characterization of Organic Compounds
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Thin layer chromatography was carried out using 60 F254 silica gel plates (Merck, Darmstadt, Germany) using appropriate solvent mixtures. Solvents were ACS reagent grade and anhydrous solvents (Sigma-Aldrich, St. Louis, Missouri, USA, and Acros Organics/Fisher Scientific, Pittsburgh, PA USA,) were used as received. Medium pressure chromatography for compound purification was carried out using Isolera with Silicycle HP cartridges (Biotage, Charlotte, NC, USA,). LCMS was performed using an 1100 HPLC system (Agilent, Santa Clara, CA, USA) equipped with a XTerra MS C18 5 µm, 4.6 Å~ 50 mm column (Waters, Milford, MA, USA ) or a Poroshell 120 EC-C18 4.6 × 100 mm column (Agilent, Santa Clara, CA, USA) using an Agilent photodiode array detector and an in-line Agilent 6130 single quadrupole mass spectrometer. Analytical HPLC method involved gradient elution from 0 to 95% acetonitrile in water (0.1% formic acid) over 6 min. Agilent ChemStation software (Agilent, Santa Clara, CA, USA) was used to develop methods. Final purity of the compounds was determined by 1H-NMR (AV-300, Brucker, Billerica, MA, USA) or by analyzing chromatogram of the products at 210, 254 and 280 nm.
5
Ginsenoside Fraction Purification
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GFRP (including 1.84%, 1.04%, 0.75%, 0.16% and 0.70% of Rb1, Rc, Rb2, Rb3 and Rd (w/w) determined by HPLC, for details see S-Fig. 2 B) was purchased from Jilin Province Caisenren Biotechnology Co., Ltd, China. Standards of 20(S)-Rg3 (lot number MUST-12041211), 20(R)-Rg3 (lot number MUST-12080811) and Rg5 (lot number MUST-15051917) were all at 98% purity and purchased from Mansite cooperation, Chengdu, China. High-Performance Liquid Chromatography (HPLC)-grade acetonitrile was purchased from Oceanpak company, Germany; HPLC-grade methanol and analytical grade 95% ethanol were purchased from Tianjin Damao company, China. HPLC solvents were filtered through 0.45 μm membrane filters prior to use. 60F254 Silica gel plates purchased from Merck, Germany.
6
Physicochemical Characterization of Compounds
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All commercial reactants and solvents with the highest purity were purchased from either Sigma-Aldrich (St. Louis, MS, USA) or Alfa Aesar (Karlsruhe, Germany) and used without further purification. The melting points were determined on a Fischer-Johns block (Fisher Scientific, Schwerte, Germany) and are uncorrected. Elemental analyses were determined by an AMZ-CHX elemental analyzer (PG, Gdańsk, Poland) and are within ±0.4% of the theoretical values. ¹H-NMR spectra were recorded on an Avance 300 MHz) spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). Analytical thin layer chromatography was performed with 60F254 silica gel plates (Merck, Darmstadt, Germany) and visualized by UV irradiation (254 nm). The physicochemical characterization of compounds 7 and 18 was presented in [43 (link),44 (link)]. The structure of 4 was presented in our previous publication [45 (link)], however no physicochemical characterization data was reported.
7
Synthesis of Clickable Lipid Conjugates
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Unless stated otherwise, all reagents and
chemicals were obtained from commercial sources at the highest purity
available and used without further purification. All solvents were
of AR quality and purchased from Biosolve. Water was purified on an
EMD Millipore Milli-Q Integral Water Purification System. Reactions
were followed by thin-layer chromatography (precoated 0.25 mm, 60-F254
silica gel plates from Merck). Dry solvents were obtained with an
MBRAUN Solvent Purification System (MB-SPS). Ion exchange resin DOWEX
1X8-50 (Cl-form) was obtained from Acros. Prior to use, a column was
first washed with demineralized water, followed by washing with methanol.
Weakly basic resin Amberlite IRA-95 (Aldrich) was washed with water,
methanol, and again water before use. Automated column chromatography
was performed on a Biotage Isolera using Biotage SNAP-KP SIL cartridges.
H2N-C12-EO4-N3,18 (link) H2N-C12-EO4-OBn,13 (link) Chol-NHS,35 (link) Atr-C5-OH*HCl,36 (link) DMT-MM,37 (link) 5-methoxycarbonyl-benzene-1,3-dicarboxylic acid
(3),38 (link) andBTA-(OH) 3 (13 (link)) were synthesized according
to previously published procedures.
chemicals were obtained from commercial sources at the highest purity
available and used without further purification. All solvents were
of AR quality and purchased from Biosolve. Water was purified on an
EMD Millipore Milli-Q Integral Water Purification System. Reactions
were followed by thin-layer chromatography (precoated 0.25 mm, 60-F254
silica gel plates from Merck). Dry solvents were obtained with an
MBRAUN Solvent Purification System (MB-SPS). Ion exchange resin DOWEX
1X8-50 (Cl-form) was obtained from Acros. Prior to use, a column was
first washed with demineralized water, followed by washing with methanol.
Weakly basic resin Amberlite IRA-95 (Aldrich) was washed with water,
methanol, and again water before use. Automated column chromatography
was performed on a Biotage Isolera using Biotage SNAP-KP SIL cartridges.
H2N-C12-EO4-N3,18 (link) H2N-C12-EO4-OBn,13 (link) Chol-NHS,35 (link) Atr-C5-OH*HCl,36 (link) DMT-MM,37 (link) 5-methoxycarbonyl-benzene-1,3-dicarboxylic acid
(3),38 (link) and
to previously published procedures.
8
Synthesis and Characterization of Compounds 5a-j
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All the melting points were determined on a Cintex melting point apparatus and are uncorrected. Analytical TLC was performed on Merck precoated 60 F254 silica gel plates. Visualization was done by exposing to iodine vapour. IR spectra (KBr pellet) were recorded on a PerkinElmer BX series FT-IR spectrometer. 1H NMR spectra were recorded on a Varian Gemini 300 MHz spectrometer. 13C NMR spectra were recorded on a Bruker 75 MHz spectrometer. Chemical shift values are given in ppm (δ) with tetramethylsilane as an internal standard. Mass spectral measurements were carried out by the EI method on a Joel JMC-300 spectrometer at 70 eV. The synthesis of compounds 5a-j was accomplished by synthetic route shown in scheme 1 .
9
Purification and Characterization of Chemical Compounds
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The chemicals
and solvents used laboratory grade and were purified
as per literature methods. All reactions have been monitored by thin
layer chromatography (TLC). TLC was performed on Merck 60 F-254 silica
gel plates. 1H NMR and 13C NMR spectra were
recorded at either 500 or 400 MHz (1H NMR) and 126 or 100
MHz (13C NMR) on the Bruker spectrometer instruments. The
HRMS spectra were recorded on the Bruker Compass Data Analysis 4.2.
The column chromatography was performed on silica gel for column chromatography
(100–200mesh) which was supplied by Thermo Fisher Scientific
India Pvt. Ltd.
and solvents used laboratory grade and were purified
as per literature methods. All reactions have been monitored by thin
layer chromatography (TLC). TLC was performed on Merck 60 F-254 silica
gel plates. 1H NMR and 13C NMR spectra were
recorded at either 500 or 400 MHz (1H NMR) and 126 or 100
MHz (13C NMR) on the Bruker spectrometer instruments. The
HRMS spectra were recorded on the Bruker Compass Data Analysis 4.2.
The column chromatography was performed on silica gel for column chromatography
(100–200mesh) which was supplied by Thermo Fisher Scientific
India Pvt. Ltd.
10
Thin Layer Chromatography of Ginsenosides
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The thin layer chromatography (TLC) was performed using 60F254 silica gel plates (Merck, Germany) with CHCl3-CH3OH-H2O (65∶35∶10, lower phase) as the solvent. The spots on the TLC plates were identified through comparisons with standard ginsenoside after visualization was made by spraying 10% (vol/vol) H2SO4, followed by heating at 110°C for 5 min.
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