Enzyme activity and substrate specificity of DGAT1 were measured in vitro by the incubation of microsomal fractions with different combinations of radiolabeled fatty acyl-CoA substrate and DAG as described previously with some modifications (Shockey et al., 2006 (link)). Briefly, 25 μg of microsomal membrane proteins were incubated with 10 μM [1- 14C] acyl-CoAs [palmitoyl (16:0) -CoA or oleoyl (18:1) -CoA from PerkinElmer, specific activity, 20,000 CPM/μmol] and 400 μM DAG [di-palmitin (di16:0) and diolein (di18:1); stock of 40 mM in methanol)] in a total reaction volume of 100 µL buffered with 100 μM Tris-HCl (pH 8.0). The reaction mixture was incubated at 37°C for one hour in a water bath. Lipids were extracted, concentrated under vacuum, and dissolved in 20 µL chloroform. Lipids were spotted on Silica Gel 60 plates (Whatman) thin layer chromatography (TLC) plates and separated as described previously (Janero and Barrnett, 1981 (link)). Developed TLC plates were dried for 10 min and placed on AR-2000 radio-TLC imaging scanner (Bioscan, Inc., USA) to detect and quantify the radioactive TAG spots. Assay mixtures including all components except microsomal proteins were used as a negative control. The in vitro assay was performed in triplicate for each acyl-CoA substrate and each enzyme. The obtained data are represented as mean ± SD.
Ar 2000 radio tlc imaging scanner
Manufactured by Bioscan
Sourced in United States, France
The AR-2000 radio-TLC Imaging Scanner is a laboratory equipment used for the detection and quantification of radioactively labeled compounds on thin-layer chromatography (TLC) plates. The device scans the TLC plate and generates an image that represents the radioactive distribution across the plate.
Automatically generated - may contain errors
Lab products found in correlation
Flexanalysis 3, by Bruker (2 mentions)
1100 series, by Agilent Technologies (2 mentions)
Silica gel 60 plates, by Cytiva (1 mentions)
Millipore filter, by Merck Group (1 mentions)
Ct 26 cells, by Korean Cell Line Bank (1 mentions)
Wizard 1470 automatic gamma counter, by PerkinElmer (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Dimethylformamide dmf, by Merck Group (1 mentions)
Balb c mice, by Orient Bio (1 mentions)
Amicon ultra centrifuge filter, by Merck Group (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
2470 wizard2, by PerkinElmer (1 mentions)
Tilmanocept, by Eppendorf (1 mentions)
Lobind, by Eppendorf (1 mentions)
Winscan software, by Bioscan (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Wizard gamma counter, by PerkinElmer (1 mentions)
Nanospect, by Bioscan (1 mentions)
Silica gel, by Agilent Technologies (1 mentions)
Ultraflex 3 tof tof, by Bruker (1 mentions)
18 protocols using ar 2000 radio tlc imaging scanner
1
DGAT1 Enzyme Activity Assay
2
Technetium-99m Nanobody Purification and Radiolabeling
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The 99mTc-nanobody solution was purified by ultrafiltration and washed twice using PBS to remove the dissociative [99mTc(H2O)3(CO)3]+. Then the 99mTc-nanobody solution was passed through a 0.22-µm Millipore filter (EMD Millipore) to eliminate possible aggregates. Thin layer chromatography (TLC) was then performed to determine the labeling efficiency and radiochemical purity of the 99mTc-nanobody, directly after labeling and after purification. The analytes were spotted on silica gel plates, which were subsequently developed in acetone and detected using an AR-2000 radio-TLC Imaging Scanner equipped with a 10% methane:argon gas supply and the running analysis software Winscan version v.3 (Bioscan Inc., Washington, DC, USA). The analysis of crude mixtures was used to calculate the yield, and the analysis of the purified product was used to calculate the radiochemical purity.
3
Radiolabeling of Organic Substrates
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Example 73
A 4 mL vial with a screw cap was charged with substrate (0.22 mmol), iodosylbenzene (0.068 mmol) and a stir bar (2×5 mm). A portion of aqueous [18F]fluoride solution (40-50 μL, 4-5 mCi) obtained from the cyclotron was loaded on to an Chromafix PS-HCO3 IEX cartridge, which had been previously washed with 5.0 mg/mL K2CO3 in Milli-Q water followed by 5 mL of Milli-Q water. Then, the cartridge loaded with [18F]fluoride was washed with 2 mL Milli-Q water and [18F]fluoride was released from the cartridge using 0.8 mL 5.0 mg/mL K2CO3 in Milli-Q water. A portion of the resulting [18F]fluoride solution (25 μL, 125-150 μCi) was diluted with 3.0 mL acetonitrile. 0.6 mL of this [18F]fluoride acetonitrile solution was added to the vial containing the substrate and the oxidant. The resulting mixture was stirred for 2 min under 50° C. (for most of the cases, PhIO solid will dissolve during the stirring). Then 2 mg Mn(TMP)Cl catalyst (0.0023 mmol) was added in solid form to the reaction mixture. The vial was recapped and stirred at 50° C. for 10 more min. After 10 min, an aliquot of the reaction mixture was taken and spotted on a silica gel TLC plate. The plate was developed in an appropriate eluent and scanned with a Bioscan AR-2000 Radio TLC Imaging Scanner.
4
Zr-89 Radiolabeled Nanoparticles for Cancer Imaging
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Tetraethyl orthosilicate (TEOS), NH4OH, (3-Aminopropyl) triethoxysilane (APTES), triphenylphosphonium bromide (TPP), folic acid (FA), 1-ethyl-3-(3-dimaethylaminopropyl) carbodiimide (EDC), N-hydroxy succinimide (NHS), triethylamine (TEA) and dimethylformamide (DMF) were purchased from Sigma-Aldrich. Dulbecco’s Modified Eagle’s Medium was purchased from Gibco BRL Life Technologies. Carcinoma cells, CT-26 cells were procured from Korean cell line bank (Seoul, Korea) and Balb/c mice (20 ± 1.5 g, female) were purchased from Orient Bio (Seongnam, Korea). 89Zr was produced using RFT-30 (30 MeV indigenous cyclotron) at Korea Atomic Energy Research Institute (KAERI). Dynamic light scattering analysis was measured using Zeta-sizer (Malvern, UK). Radioactivity was measured using a CRC-15R ionizing chamber (Capintec, Florham Park, NJ, USA). Radiochemical yield was assessed using an AR-2000 radio-TLC imaging scanner (Bioscan, Santa Barbara, CA, USA). The cell uptake of the radiolabeled SNPs was measured with a Wizard-1470 automatic gamma counter (Perkin Elmer, Waltham, MA, USA). Small animal PET imaging was carried out using a Genesis 4 (Sofie Biosciences, Culver City, CA, USA) machine.
5
Synthesis and Evaluation of DTPA-AHNP-PEG Conjugate
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For preparation of DTPA conjugated-AHNP-PEG (DTPA-AHNP-PEG), AHNP-PEG and p-SCN-Bn-DTPA (w/w 1:50) was soaked in sodium carbonate buffer at 25 °C for 8 h. The conjugated-compounds were then purified by Amicon ultra centrifuge filters (3 kDa) (Merck Millipore). For molecular weight measurement, AHNP-PEG and DTPA-AHNP-PEG were detected with the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Billerica, MA, USA). The spectra were processed using FlexAnalysis™ 3.0 software (Bruker Daltonics). The DTPA-AHNP-PEG conjugation efficiency with indium-111 (111In-DTPA-AHNP-PEG) was evaluated by instant thin layer chromatography (ITLC) (AR-2000 radio-TLC Imaging Scanner; Bioscan, Washington, DC, USA).
6
Purification and Characterization of 125I-RGD4Cβl
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The crude product was transferred to a chromatography column filled with Sephadex G-50, which was pre-blocked by bovine serum albumin, and eluted by PB buffer (pH=7.4). The effluent was collected in tubes. The radioactivity in each tube was measured with a γ counter, (2470 WIZARD2, PerkinElmer, Inc., Waltham, MA, USA), and tubes 14–17 were collected. The final product, 125I-RGD4CβL, was obtained and passed through a 0.22 µm Millipore filter to eliminate possible aggregates. Thin-layer chromatography (TLC) was then performed using an AR-2000 radio-TLC Imaging Scanner, Bioscan (Washington, DC, USA) to determine the labeling efficiency and the radiochemical purity of the 125I-RGD4CβL, directly subsequent to labeling and purification, using acetone as a developing solvent.
7
Radiolabeling of 99mTc-3P4-RGD2 Conjugate
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The 99mTc-3P4-RGD2 conjugate was generously provided by the Medical Isotopes Research Center of Peking University (Beijing, China) as a freeze-dried kit. 99mTc-3P4-RGD2 was labeled with Na99mTcO4 (China Institute of Atomic Energy, Beijing, China) solution, followed by 20-min incubation at 100°C. Quality control was conducted using radioactive thin-layer chromatography (using an AR-2000 radio-TLC Imaging Scanner, Bioscan, Inc., Washington, DC, USA), which enabled to measure a high labeling yield of ~95%. A molybdenum-technetium generator was provided by Beijing Atom Hi-Tech Co., Ltd. (Beijing, China).
8
Radiolabeling and Quality Control of Tilmanocept
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Prior to its radiolabeling, 62.5 µg of Tilmanocept (Lymphoseek®, Norgine BV, Amsterdam, The Netherlands) were reconstituted with 200 µL of NaCl 0.9%. Then, a given volume of sodium pertechnetate (99mTcO4−, freshly “milked” from a Tekcis® generator, Curium, Saclay, France) was transferred into a Lobind® (Eppendorf, Hambourg, Germany) microtube with Tilmanocept in order to obtain 2350 ng of Tilmanocept in a final volumic activity of 150 MBq.mL−1. The microtube was then placed under agitation at 1000 rpm for 15 min at 25 °C.
The radiochemical purity was checked by radio-ITLC (Instant Thin Layer Chromatography) using a γ-radiochromatograph (AR-2000 Radio-TLC & Imaging Scanner, Bioscan, Washington, DC, USA). Briefly, 1μL of solution was deposited on cellulose chromatographic paper (Whatman Grade 1, 3MM, 31ET Chr, Merck, France) and eluted with acetone to separate the 99mTcO4− (“free” Tc-99m) from 99mTc linked to Tilmanocept. On the chromatographic strip, the 99mTc-Tilmanocept remains at the deposition point while the “free” Tc-99m migrates to the solvent front. The radiochromatograph obtained allows determination of the radiochemical purity by integration of the different peaks. Results were obtained using the WinScan® software (Bioscan, Washington, DC, USA).
The radiochemical purity was checked by radio-ITLC (Instant Thin Layer Chromatography) using a γ-radiochromatograph (AR-2000 Radio-TLC & Imaging Scanner, Bioscan, Washington, DC, USA). Briefly, 1μL of solution was deposited on cellulose chromatographic paper (Whatman Grade 1, 3MM, 31ET Chr, Merck, France) and eluted with acetone to separate the 99mTcO4− (“free” Tc-99m) from 99mTc linked to Tilmanocept. On the chromatographic strip, the 99mTc-Tilmanocept remains at the deposition point while the “free” Tc-99m migrates to the solvent front. The radiochromatograph obtained allows determination of the radiochemical purity by integration of the different peaks. Results were obtained using the WinScan® software (Bioscan, Washington, DC, USA).
9
Radiolabeling of Human Serum Albumin
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Example 1
The type of human serum albumin used in the present invention was SK albumin 20% (SK Chemicals, Korea), and the PD-10 column (GE) was used for purification of reaction mixture. The radioactive isotope [Tc-99m]TcO4− used in this invention was purchased from Ultra-Technekow DTE Generator (Covidien). Instant thin layer chromatography (ITLC) that was used to identify labeling was purchased from Pall Life Science. TLC scanning was performed using AR-2000 radio-TLC Imaging Scanner (Bioscan). Also, the type of gamma counter used in a biodistribution experiment was the Wizard gamma counter (Perkin Elmer). BCA protein assay kit and Ellman's reagent were purchased from Pierce, and other reagents and solvents were purchased from Sigma-Aldrich. Gamma images of animals were obtained by using NanoSPECT (Bioscan) and near infrared region (NIR) fluorescent images were obtained by using IVIS Lumina (Xenogen).
10
Evaluating In Vitro Stability of 111In-DOTA-EB-cRGDfK
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In vitro stability of 111In-DOTA-EB-cRGDfK was evaluated by incubation with normal saline (in 1:1 vol ratio) or rat plasma (in 1:19 vol ratio) at room temperature. The radiochemical purity was determined by iTLC analysis as previously described [43 (link)]. At desired times (0, 2, 4, 24, 48, 72, and 96 h), 1 μL was using for iTLC on the glass microfiber chromatography paper impregnated with a silica gel (Agilent Technologies, Santa Clara, CA, USA), whereas the mobile phase was used as 0.1 M citric acid/0.1 M sodium citrate (v/v = 2.1/7.9) buffer. Then, the sheets were measured using a radioactive scanner (AR-2000 radio-TLC Imaging Scanner, Bioscan, France).
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