124I was purchased from IBA Molecular, VA, USA (2.6 TBq/mL, n.c.a.). Radio-iodination (nucleophilic aromatic addition) was performed by adding Na124I (2.6 TBq/mL, no carrier added) into TAT-ΔNS3/4A-FITC (0.5 mg, MW = 4339.8) with hydrogen peroxide solution (H2O2: HCl: H2O = 16:16:68) as an oxidant under condition. The solution was reacted at room temperature for 10 min with vigorous vortexing, followed by adding 2 mM sodium thiosulfate solution and saturated sodium hydrogen carbonate to neutralize the solution. TAT-ΔNS3/4A-124I-FITC was purified by C-18 sep-pak (Waters, Milford). The radiochemical purity of TAT-ΔNS3/4A-124I-FITC was determined using HPLC (Delta 600, Waters). For HPLC analysis, a reverse phase column (Purospher STAR RP-18e, 10 × 250 mm, MERCK, Darmstadt, Germany) was used and eluted with acetonitrile (ACN)/0.1% TFA in water (3/97, v/v) at a flow rate of 4 mL/min.
Purospher star rp 18e
Manufactured by Merck Group
Sourced in Germany
Purospher STAR RP-18e is a reversed-phase high-performance liquid chromatography (HPLC) column produced by Merck. It features a silica-based stationary phase with octadecyl (C18) functional groups. The column is designed for the separation and analysis of a wide range of organic compounds.
Automatically generated - may contain errors
Lab products found in correlation
Delta 600, by Waters Corporation (1 mentions)
Sep pak c18, by Waters Corporation (1 mentions)
Lichrocart precolumn, by Merck Group (1 mentions)
Inova 400m nmr spectrometer, by Agilent Technologies (1 mentions)
Agilent 1200 hplc system, by Agilent Technologies (1 mentions)
Dna extraction kit, by Fujifilm (1 mentions)
6 protocols using purospher star rp 18e
1
Radiolabeling of TAT-ΔNS3/4A-FITC with I-124
2
Analytical Characterization of Organic Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All
reagents and solvents
were of commercial quality and used without further purification. 1H and 13C NMR data were recorded on a Varian Inova
400 M NMR spectrometer operating at 400 and 101 MHz for 1H and 13C, respectively. All chemical shifts were in ppm
(δ) with respect to tetramethylsilane (TMS) as an internal standard,
and coupling constants (J) were in Hz. Mass spectra were obtained
on DSQ (low-resolution mass spectrometer) and MAT95XP (high-resolution
mass spectrometer) instruments. The purities (>95%) of all target
compounds were checked by HPLC using an LC-2010c with a UV detector.
Samples were injected on a Merck Purospher STAR RP-18e 125 cm 4.6
mm (5 μm) column equipped with a Merck Lichrocart precolumn
(Merck, Germany). Analyses were run according to a solvent system
(see below). The traces were recorded at 254 nm, and the column temperature
was 40 °C. The solvent system consists of a binary system with
0.1% (v/v) aqueous formic acid and acetonitrile 20:80 (v/v), with
a flow rate of 0.8 mL/min.
NMR and X-ray data are given in theSupporting Information .
reagents and solvents
were of commercial quality and used without further purification. 1H and 13C NMR data were recorded on a Varian Inova
400 M NMR spectrometer operating at 400 and 101 MHz for 1H and 13C, respectively. All chemical shifts were in ppm
(δ) with respect to tetramethylsilane (TMS) as an internal standard,
and coupling constants (J) were in Hz. Mass spectra were obtained
on DSQ (low-resolution mass spectrometer) and MAT95XP (high-resolution
mass spectrometer) instruments. The purities (>95%) of all target
compounds were checked by HPLC using an LC-2010c with a UV detector.
Samples were injected on a Merck Purospher STAR RP-18e 125 cm 4.6
mm (5 μm) column equipped with a Merck Lichrocart precolumn
(Merck, Germany). Analyses were run according to a solvent system
(see below). The traces were recorded at 254 nm, and the column temperature
was 40 °C. The solvent system consists of a binary system with
0.1% (v/v) aqueous formic acid and acetonitrile 20:80 (v/v), with
a flow rate of 0.8 mL/min.
NMR and X-ray data are given in the
3
HPLC Analysis of Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The solvents used in high performance liquid chromatography are of analytic grade from Merck®. A Milli-Q System® (Bedford, MA, USA) was used to purify the water. Analyses of high performance liquid chromatography was performed on a Merck-Hitachi liquid chromatograph LaChrom Elite® equipped with a VRW HITACHI L- 2130 pump, a VRW HITACHI L-2300 Diode-Array Detector (DAD), and an auto sampler with a 100 μl loop. The data was acquired and processed using Ezchrom Elite software. The extract was analyzed using a reverse-phase HPLC column: Purospher® STAR RP-18e (250 mm × 4.6 mm i.d., 5 μm) column (Merck). The mobile phase was composed of solvent (A) H2O/H3 PO4 0.1% and solvent (B) MeOH. The solvent gradient was composed of A (75-0%) and B (25-100%) for 20 minutes, then 100% B for 4 minutes, then again at the initial conditions (75% A and 25% B) for 10 minutes. A flow rate of 1.0 ml/minutes was used in a 30°C oven, and 20 μl of each sample was injected. The procedure was repeated three times for each sample. Samples and mobile phases were filtered through a 0.22 μm Millipore filter prior to HPLC injection. Spectra data were recorded from to 200 to 400 nm during the entire run.
4
HPLC Analysis of Phenolic Acids and Flavonoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A new HPLC method was developed for the detection of phenolic acids and flavonoids by employing an HPLC system (Waters 2695, Milford, MA, USA) equipped with a photodiode array detector (Waters 2996). The HPLC column was a Merck Purospher Star, RP-18e (150 × 4.6 mm, 5 μm), fitted with a guard cartridge that had been packed with a similar type of stationary phase (Merck). A linear gradient flow was employed at a flow rate of 0.5 mL/min throughout and the total analytical time was approximately 40 min.
The binary mobile phase consisted of solvent A (ultrapure water with 0.1% phosphoric acid) and solvent B (pure methanol with 0.1% phosphoric acid). Elution from the column was achieved with the following gradients: 0 to 20 min of solvent B, increasing from 10% to 85%; 20 to 25 min of solvent B at 85% throughout; 25 to 26 min of solvent B from 85% decreasing to 10%; and a final composition at 10% that was kept constant to 40 min. The detection wavelength was set between 200 and 450 nm, with specific monitoring conducted at 220 nm. Identification of the phenolic and flavonoid compounds was performed by comparing the retention times of the analytes with reference standards.
The binary mobile phase consisted of solvent A (ultrapure water with 0.1% phosphoric acid) and solvent B (pure methanol with 0.1% phosphoric acid). Elution from the column was achieved with the following gradients: 0 to 20 min of solvent B, increasing from 10% to 85%; 20 to 25 min of solvent B at 85% throughout; 25 to 26 min of solvent B from 85% decreasing to 10%; and a final composition at 10% that was kept constant to 40 min. The detection wavelength was set between 200 and 450 nm, with specific monitoring conducted at 220 nm. Identification of the phenolic and flavonoid compounds was performed by comparing the retention times of the analytes with reference standards.
5
HPLC Analysis of Phytocompounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extracts were chemically characterized on an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA) with a diode array detector. Purospher STAR RP-18e (150 × 4.6 mm) with the particle size of 5 µm (Merck, Darmstadt, Germany) was used as the analytical column. The extracts were primarily dissolved in ultrapure methanol, HPLC grade (10 mg/mL), and filtered through a 0.45 µm microfilter. The extract solutions were injected at a volume of 10 µL. The mobile phase consisted of a mixture of 0.1% aqueous trifluoroacetic acid (A) and acetonitrile (B) with a linear gradient: 0–3 min 5–5% B, 3–32 min 5–28% B, 32–44 min 25–50% B, 44–52 min 50–80% B, 52–54 min 80–90% B, 54–59 min 90–5% B, and 59–60 min 5% B. The flow in the column was 0.7 mL/min and the operating temperature was maintained at 30 °C. Phytocompounds were identified and quantified on the basis of UV-Vis signal response compared to standards. Their quantities in the extracts were expressed as µg/mg [24 (link)].
6
Quantifying 8-OHdG in DNA Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
8-OHdG formation was determined using an HPLC-ECD system according to the method of Asami et al. [27 (link)]. After each exposure to UV irradiation, calf thymus DNA was isolated from the reaction mixture using a DNA-extraction kit (Wako, Osaka, Japan) according to the manufacturer’s protocol, with minor modifications to prevent the formation of 8-OHdG during DNA isolation. Isolated DNA was then digested with nucleases to obtain 8-OHdG in the nucleoside form, after which the nucleosides were injected into a Purospher® STAR RP-18e (5 μm, 4.0 × 250 nm, Merck Chemicals, Darmstat, Germany) connected to an HPLC system. The latter system consisted of a HITACHI (Tokyo, Japan) L-2130 pump and a UV 7000 detector (EYELA, Tokyo, Japan). Electrochemical detection was accomplished using an ECD (Coulochem® III, Guard Cell 5020; ESA Inc., Dionex, Tokyo, Japan). The mobile phase consisted of 0.2 M Na2PO4 containing 6% methanol. The flow rate was 1.0 mL/min with the following applied conditions: E1: 150 mV, R: 1 μA, Filter: 10 s, output: 1.0 V, E2: 300 mV, R: 50 μA, Filter: 10 s, and output: 1.0 V. DNA-specific 8-OHdG was expressed in terms of the ratio of 8-OHdG to deoxyguanosine (2dG).
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!