Cerebrospinal fluid sample collection and its analysis was performed as described previously in (9 (link)). Briefly, CSF was centrifuged immediately after collection (1600 g, 4 °C, 15 minutes), aliquoted into polypropylene test tubes (each aliquot, 750 µL), frozen within 30–40 minutes after the puncture and stored at −80 C until use. After thawing, 700 µL of CSF were diluted with 700 µL buffer (pH 10.5; 2 M urea, 100 mmol/L NaCl, 0.0125% NH3) and ultracentrifuged using Centrisart ultrafiltration devices (cut off 20 kDa) at 4 °C until 1.1 mL of filtrate was obtained. Subsequently the filtrate was applied onto a PD-10 desalting column (Amersham Biosciences, Freiburg, Germany) equilibrated in 0.01% NH4OH in HPLC-grade water (Roth, Karlsruhe, Germany), and the eluate was lyophilized, stored at 4 °C and resuspended in 10 µL HPLC-grade water before capillary electrophoresis–mass spectrometry (CE-MS) analysis. CE-MS analysis was performed as described using a P/ACE MDQ (Beckman Coulter, Krefeld, Germany) system on-line coupled to a Micro-TOF MS (Bruker Daltonic, Bremen, Germany) (9 (link), 22–24 (link)).
Hplc grade water
Manufactured by Carl Roth
Sourced in Germany
HPLC-grade water is a highly purified form of water specifically designed for use in high-performance liquid chromatography (HPLC) applications. It has low levels of impurities and contaminants, ensuring consistent and reliable performance in HPLC systems.
Automatically generated - may contain errors
Lab products found in correlation
Microtof ms, by Bruker (2 mentions)
P ace mdq, by Beckman Coulter (1 mentions)
Pd 10 desalting column, by Cytiva (1 mentions)
Chloroform, by Carl Roth (1 mentions)
Ms grade water, by Carl Roth (1 mentions)
Methanol, by Carl Roth (1 mentions)
Acetonitrile, by Carl Roth (1 mentions)
Spermidine, by Merck Group (1 mentions)
Netropsin, by Merck Group (1 mentions)
Primus 25 advanced, by Avantor (1 mentions)
Ferritin from equine spleen, by Merck Group (1 mentions)
Zepto, by Diener Electronic (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Sprayer, by Agilent Technologies (1 mentions)
P ace mdq capillary electrophoresis system, by Beckman Coulter (1 mentions)
Formic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Avantor (1 mentions)
6 protocols using hplc grade water
1
Cerebrospinal Fluid Sampling and Analysis
2
Metabolite Quantification with Stable Isotopes
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MS-grade water and acetonitrile for the preparation of mobile phases and samples were purchased from Carl Roth (Karlsruhe, Germany). Metabolite standards and reagents (>99% p.a) were purchased from Sigma-Aldrich (Schnelldorf, Germany). The standard stock solutions were prepared in MS-grade water and stored at −70 °C. [U13C]-d -glucose (99 atom%) for the preparation of fully labeled cellular extract for IDMS was purchased from Silantes (München, Germany). [U13C]- ( 99 atom%) and [1-13C1]-d -glucose (99 atom%) for isotopic tracer experiments were purchased from Cambridge Isotope Laboratories (Tewksbury, USA) and Sigma-Aldrich (Schnelldorf, Germany), respectively. Chloroform (>99% p.a.), HPLC-grade water, and methanol for quenching and extraction was purchased from Carl Roth (Karlsruhe, Germany) and VWR chemicals (Darmstadt, Germany), respectively.
3
Investigating MB Binding to DNA Origami
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For
the initial experiments
addressing the binding mode of MB, lyophilized genomic dsDNA from
salmon testes (Alfa Aesar) was dissolved at the desired concentration
either in high-performance liquid chromatography (HPLC)-grade water
(Carl Roth) or in 1× TAE (Calbiochem) with 10 mM MgCl2 (Sigma-Aldrich). MB, spermidine, and netropsin (all Sigma-Aldrich)
were added to achieve the desired concentrations.
For comparison
with the DNA origami nanostructures, synthetic dsDNA was prepared
by hybridization of two complementary oligonucleotides (Metabion)
with the sequences 5′-TTG GAA CAG CAT TGA-3′ and 5′-TCA
ATG CTG TTC CAA-3′. To this end, the readily mixed sample (100
μM of each strand in 1× TAE with 10 mM MgCl2) was heated to 94 °C in a thermocycler Primus 25 advanced (PEQLAB),
kept at this temperature for 5 min, and cooled down to 20 °C
with a cooling rate of 1.5 °C per minute.
the initial experiments
addressing the binding mode of MB, lyophilized genomic dsDNA from
salmon testes (Alfa Aesar) was dissolved at the desired concentration
either in high-performance liquid chromatography (HPLC)-grade water
(Carl Roth) or in 1× TAE (Calbiochem) with 10 mM MgCl2 (Sigma-Aldrich). MB, spermidine, and netropsin (all Sigma-Aldrich)
were added to achieve the desired concentrations.
For comparison
with the DNA origami nanostructures, synthetic dsDNA was prepared
by hybridization of two complementary oligonucleotides (Metabion)
with the sequences 5′-TTG GAA CAG CAT TGA-3′ and 5′-TCA
ATG CTG TTC CAA-3′. To this end, the readily mixed sample (100
μM of each strand in 1× TAE with 10 mM MgCl2) was heated to 94 °C in a thermocycler Primus 25 advanced (PEQLAB),
kept at this temperature for 5 min, and cooled down to 20 °C
with a cooling rate of 1.5 °C per minute.
4
Fabrication and Ferritin Immobilization on Al2O3 Substrates
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The flat and nanofaceted Al2O3 substrates were cleaned through incubation in 2% Hellmanex solution (Hellma GmbH & Co. KG, Müllheim, Germany) for 1 h at room temperature, washing with HPLC-grade water (Carl Roth GmbH, Karlsruhe, Germany), and drying in a stream of argon. Finally, the substrates were treated for 30 s with an oxygen plasma (diener Zepto, Diener electronic, Ebhausen, Germany) to remove any remaining organic contaminations.
Ferritin from equine spleen (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was prepared at concentrations of 10 mg/mL and 30 mg/mL in PBS (pH 7.4, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Furthermore, 200 µL of the prepared protein solution was carefully placed on each substrate surface and incubated for 5 h at room temperature in an environmental chamber. After incubation, the substrates were rinsed with 1 mL of HPLC-grade water and dried in a stream of argon.
Ferritin from equine spleen (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was prepared at concentrations of 10 mg/mL and 30 mg/mL in PBS (pH 7.4, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Furthermore, 200 µL of the prepared protein solution was carefully placed on each substrate surface and incubated for 5 h at room temperature in an environmental chamber. After incubation, the substrates were rinsed with 1 mL of HPLC-grade water and dried in a stream of argon.
5
Capillary Electrophoresis-Mass Spectrometry Protocol
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Capillary electrophoresis coupled to mass spectrometer (CE-MS) analysis was carried out as described by Theodorescu et al. 2006 [15 (link)]. A P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Brea, CA) being linked online to a micro-TOF MS (Bruker Daltonik, Leipzig, Germany) was used. The sprayer (Agilent Technologies, Santa Clara, CA) interfacing the CE and MS was grounded. 256 nl of the sample was injected hydrodynamically on an untreated silica capillary (New Objective, Woburn, USA, 90 cm x 50 μm). A solution of 20% acetonitrile (Sigma-Aldrich, Taufkirchen, Germany) in HPLC-grade water (Roth, Karlsruhe, Germany) supplemented with 0.94% formic acid (Sigma-Aldrich) was used as running buffer. Interface potential was adjusted to -4.5 kV. The capillary temperature was held at 35°C. Mass spectra were recorded for three seconds with signals at an m/z range between 350 and 3000. The detection limit of the TOF-Analyzer was 1 fmol [15 (link)].
6
Adsorption of Human Serum on Surfaces
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Heat-inactivated human serum from male AB clotted whole blood (product number H5667, Sigma-Aldrich, St. Louis, MO, USA) was diluted in aerated phosphate buffered saline (PBS, VWR International GmbH, Darmstadt, Germany) to obtain the desired concentration (1% or 10%). The pH of the PBS was adjusted between pH 6 and pH 8 using HCl (35%) and NaOH (>99%). Serum dilutions were prepared fresh each day to ensure comparability of the results. The different serum samples (100 µL) were deposited on the different sample surfaces (1 × 1 cm2) and incubated for 2 h at room temperature. After incubation, the sample surfaces were rinsed with ca. 3 mL HPLC-grade water (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and dried in a stream of ultra-pure air.
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