Plasma samples were thawed at 4°C in a cold room. Four hundred μL of saline solution (NaCl 0.9% in 10% D2O) was added to 200 μL of each plasma sample. The mixtures were vortexed for 1 minute and centrifuged at 16, 000 g for 15 min at 4°C and 550 μL of supernatant was transferred into 5 mm Bruker NMR tubes (Z105684 Bruker 96 well rack) using Gilson 215 Liquid Handler (Trilution software version 2.0). All 1H-NMR spectra were collected on a 600 MHz Avance II NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a 5 mm CryoProbe. A Bruker sampleJet operated by IconNMR in Topspin was used to record spectra automatically. 1D CPMG-presaturated spectra for plasma were recorded. Optimal probe tuning and matching, 90° pulse length, water offset, and receiver gain were adjusted on the representative sample. The probe was automatically locked to H2O+D2O (90%+10%) and shimmed for each sample. All NMR data were acquired at 300 K.
215 liquid handler
Manufactured by Gilson
Sourced in United States
The Gilson 215 liquid handler is a versatile and reliable automated pipetting system designed for laboratory applications. It offers precise and consistent liquid handling capabilities, with a wide volume range and high accuracy. The 215 liquid handler is a tool that can be utilized in various laboratory workflows, enabling efficient and accurate liquid transfer and sample preparation tasks.
Automatically generated - may contain errors
Lab products found in correlation
Cryoprobe, by Bruker (2 mentions)
Unipoint lc software, by Gilson (2 mentions)
Aviii 600 spectrometer, by Bruker (2 mentions)
Avance 2 nmr spectrometer, by Bruker (1 mentions)
Samplejet, by Bruker (1 mentions)
717 plus autosampler, by Waters Corporation (1 mentions)
510 pump, by Waters Corporation (1 mentions)
Luna c18 column, by Phenomenex (1 mentions)
Micro cryoprobe, by Bruker (1 mentions)
Avance 600, by Bruker (1 mentions)
Avance 500, by Bruker (1 mentions)
717 autosampler, by Waters Corporation (1 mentions)
Unity 500 spectrometer, by Agilent Technologies (1 mentions)
Mat95xp, by Thermo Fisher Scientific (1 mentions)
Ivdr platform, by Bruker (1 mentions)
Dmso d6, by Cambridge Isotopes (1 mentions)
Av 400, by Bruker (1 mentions)
Aviii 600, by Bruker (1 mentions)
Esquire 2000 mass spectrometer, by Bruker (1 mentions)
Kg60 f254, by Merck Group (1 mentions)
11 protocols using 215 liquid handler
1
NMR Metabolite Profiling of Plasma
2
Instrumentation Setup for Natural Product Library
3
NMR Spectroscopy of RDPheH Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1H–15N HSQC spectra were routinely collected
at 300 K on a Bruker Avance 600 spectrometer using a 5 mm TXI (1H/13C/15N) CryoProbe with z-axis pulsed field gradients. NMR samples were prepared in buffer
A [50 mM phosphate, 100 mM NaCl, 1 μM leupeptin, and 1 μM
pepstatin A (pH 8)] and 5% D2O. A pH of 8 was selected
despite the loss of some signals because both RDPheH and RDPheH25–117 precipitate too rapidly at pH <8 for NMR analyses,
consistent with a calculated pI value of 6.8. NMR screening for formation
of the RDPheH25–117 dimer was performed at 300 K
on a Bruker Avance 500 spectrometer equipped with a SampleJet sample
changer and a 1.7 mm TCI (1H/13C/15N) Micro-CryoProbe. NMR samples for screening were made using a Gilson
215 Liquid Handler. 15N-labeled RDPheH25–117 (20 μL of 800 μM monomer in buffer A with 20% D2O) was mixed with an equal volume of a solution of the compound
of interest [20 or 100 mM in buffer A (pH 8)], and 36 μL of
the mixture was transferred to a 1.7 mm Sample Jet tube. All spectra
were processed using NMRPipe31 (link) and analyzed
using NMRView.32 (link)
at 300 K on a Bruker Avance 600 spectrometer using a 5 mm TXI (1H/13C/15N) CryoProbe with z-axis pulsed field gradients. NMR samples were prepared in buffer
A [50 mM phosphate, 100 mM NaCl, 1 μM leupeptin, and 1 μM
pepstatin A (pH 8)] and 5% D2O. A pH of 8 was selected
despite the loss of some signals because both RDPheH and RDPheH25–117 precipitate too rapidly at pH <8 for NMR analyses,
consistent with a calculated pI value of 6.8. NMR screening for formation
of the RDPheH25–117 dimer was performed at 300 K
on a Bruker Avance 500 spectrometer equipped with a SampleJet sample
changer and a 1.7 mm TCI (1H/13C/15N) Micro-CryoProbe. NMR samples for screening were made using a Gilson
215 Liquid Handler. 15N-labeled RDPheH25–117 (20 μL of 800 μM monomer in buffer A with 20% D2O) was mixed with an equal volume of a solution of the compound
of interest [20 or 100 mM in buffer A (pH 8)], and 36 μL of
the mixture was transferred to a 1.7 mm Sample Jet tube. All spectra
were processed using NMRPipe31 (link) and analyzed
using NMRView.32 (link)
4
LC-MS Based Purification and Characterization
5
Quantitative PBDE Measurement in Maternal Serum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Maternal serum samples from each subject were prepared for shipment using Center for Disease Control (CDC)’s protocols and were sent to the CDC in Atlanta for PBDE measurement using an established and validated PBDE assay. The analytical method and quality control procedures have been described previously [37 (link)]. The method used for sample processing included automatic fortification of the samples with internal standards as well as addition of formic acid and water for denaturation and dilution of the samples using a Gilson 215 liquid handler (Gilson Inc.; Middleton, WI). The samples were thereafter extracted by solid phase extraction (SPE) using a Rapid Trace (Caliper Life Sciences; Hopkinton, MA) modular SPE system. Removal of co-extracted lipids was performed on a silica/sulfuric acid column using the Rapid Trace equipment for automation. Final analytical determination of the target analytes was performed by gas chromatography isotope dilution high resolution mass spectrometry (GC-IDHRMS) employing a MAT95XP (ThermoFinnigan MAT, Bremen, Germany) instrument. The samples were analyzed for 10 PBDE congeners: PBDE-17, -28, -47, -66, -85, -99, -100, -153, -154, -183 (comprising tri-, tetra-, penta-, hexa- and hepta-brominated congeners, but not deca).
6
Serum Lipoprotein NMR Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NMR spectroscopy was used to quantify lipoprotein (sub)class concentration and composition. The sample preparation was carried out to produce the buffer composition/pH and sample-to-buffer mixing ratio requirements for Bruker IVDr platform. Serum samples were thawed at room temperature. 110 μL of 75 mM disodium phosphate buffer in H 2 O/D 2 O (80/20) with a pH of 7.4 containing 6.15 mM NaN 3 and 4.64 mM sodium 3-[trimethylsilyl] d4-propionate (Cambridge Isotope Laboratories) was pipetted into a Ritter 96 well plate. Next, 110 μL of serum was added to the buffer and mixed by aspirating and dispensing three times. Using a modified Gilson 215 liquid handler, 190 μL of each sample was transferred into 3-mm NMR SampleJet tubes. Subsequently the tubes were closed by inserting POM balls into the caps and transferred to the SampleJet autosampler where they were kept at 6 °C while queued for acquisition.
7
NMR Assay for MnaA Enzymatic Inhibition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six hundred individual compounds were selected from phenotypic screening hits for follow-up evaluation using a NMR functional assay to test for the percent inhibition of MnaA enzymatic activity. NMR assays were performed in a standard 96-well plate. Each well in columns 2-12 was filled manually with 300 µL of D 2 O buffer containing 200 nM MnaA. Column 1 was filled with 300 µL of buffer only and was used as a negative control. One microliter of 100% d6-DMSO (Cambridge Isotope Laboratories) was added to columns 1 and 12 (wells A1-H1 and A12-H12). A Hamilton ML Star Line was used to add 1.00 µL of compound to the remaining wells (A2-H11) and pipetting up and down three times to mix reagents thoroughly (final concentrations are 20 µM compound, 0.2% d6-DMSO). The enzyme reactions were initiated by adding 200 µL of UDP-ManNAc to each well (final concentration 50 µM), and the plate was shaken for 2 min. After 1 h incubation, the reactions were stopped by heating. The contents of each well were then transferred from the 96-well plate to a 96-rack NMR tube using a Gilson 215 liquid handler.
8
HPLC-SPE-NMR Workflow for Compound Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the HPLC separation, the eluate was added water by a make-up pump with a flow rate of 1.2 mL min−1, and each compound peak was passed through an individual HySphere-Resin GP cartridge (10 × 2 mm, 10–12 μm) in the Prospekt 2 automated solid-phase extraction unit. This HPLC-SPE process was repeated six to seven times. The concentration of each sample and the volume of injection per HPLC run are shown in supplementary data (Figs. S1–3). Each compound loaded cartridge was flushed with dry nitrogen stream for 30 min to remove the eluent residue and the trapped compound in the cartridge was transferred into a 2-mm NMR tube with CD3OD by a Gilson Liquid Handler 215 automated tube transfer (TT) system. The NMR tubes were then placed on an automatic NMR tube exchanging system to record NMR spectra by a Bruker AV III-600 spectrometer using a multiple solvent suppression pulse program at 298 K.
9
NMR and HPLC-MS Characterization of Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Optical rotations were obtained on a JASCO P-2000 polarimeter (Hachioji, Tokyo). UV spectra (MeOH) were measured on a Hitachi U-2900 double-beam spectrophotometer (Hitachi, Japan). Electron circular dichroic (CD) spectra (MeOH) were measured on a JASCO J-720 spectropolarimeter (Hachioji, Tokyo). NMR spectra were recorded by Bruker AV-400, and AV III-600 (CD3OD, δH 3.30 and δC 49.0 ppm). HPLC-SPE-NMR (600 MHz), composed of an Agilent 1100 liquid chromatograph (Waldbronn, Germany), a Phenomenex Prodigy ODS3 (C-18) 100 Å (250 × 4.6 mm, 5 μm) column, coupled with a diode array detector (DAD, G1315A) and a Knauer K120 HPLC pump (makeup pump), a Prospekt 2 automated solid-phase extraction unit (Spark Holland, Emmen, Holland), containing 192 HySphere resin GP cartridges (10 × 2 mm, 10–12 μm), connecting to a Gilson Liquid Handler 215 automated tube transfer (TT) system (Gilson, Inc., Middleton, WI, USA), and a Bruker AV III-600 spectrometer. HPLC-ESIMS (electrospray ionization mass spectrometry) was performed on an Agilent 1100 series liquid chromatograph, followed by an Esquire 2000 mass spectrometer (Bruker Daltonics, Germany). TLC analysis was carried out on silica gel plates (KG60-F254, Merck).
10
NMR-based Metabolomic Profiling of Serum Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NMR analyses of the metabolic measures was carried out at the University of Eastern Finland quantifying 149 metabolites from serum samples of the participants. The process has been described elsewhere
12 (link). Briefly, the samples are prepared automatically with a Gilson Liquid Handler 215, whereby 300μl of sodium phosphate NMR buffer are mixed with 300μl of serum sample. Once prepared the samples are inserted into the SampleJet™ (Bruker BioSpin GmbH, Germany) sample changer. Finally, the data are measured using a Bruker AVANCE III spectrometer. Metabolite data contains known risk factors for CAD, such as LDL-cholesterol, but also many other metabolites, as well as multiple lipoprotein subclasses. Due to the unreliability of the signal, pyruvate was removed from the analyses, leaving 148 metabolites. All abbreviations of metabolites used can be found in
Supplementary Table 2 .
12 (link). Briefly, the samples are prepared automatically with a Gilson Liquid Handler 215, whereby 300μl of sodium phosphate NMR buffer are mixed with 300μl of serum sample. Once prepared the samples are inserted into the SampleJet™ (Bruker BioSpin GmbH, Germany) sample changer. Finally, the data are measured using a Bruker AVANCE III spectrometer. Metabolite data contains known risk factors for CAD, such as LDL-cholesterol, but also many other metabolites, as well as multiple lipoprotein subclasses. Due to the unreliability of the signal, pyruvate was removed from the analyses, leaving 148 metabolites. All abbreviations of metabolites used can be found in
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!