The derivative was isolated from a development batch of insulin lispro substance by repetitive ion exchange chromatography (IEC) using ICS-5000 system with a PDA detector (Dionex, Sunnyvale, USA). 0.1 mg of the insulin lispro containing 0.8% of the derivative was injected on a DNA Pac PA-100, 250 × 4.0 column (Dionex, Sunnyvale, USA). The separation was carried out at 35°C with a linear gradient elution from 0% to 25% eluent B in 30 min at the flow rate 0.5 ml/min. Eluent A was 8 mM phosphate buffer, 67% ethanol pH 7.6 and eluent B was 0.3 M NaCl, 8 mM phosphate buffer, 67% ethanol pH 7.6. The peak of the derivative (relative retention time 0.8; see Fig. 3 ) was collected. This procedure was repeated several times to obtain sufficient amount of the derivative. All fractions of the derivative were pooled and evaporated to dryness using Concentrator Plus vacuum centrifuge (Eppendorf, Hamburg, Germany) and kept at −20°C until use.
Vacuum concentrator plus
Manufactured by Eppendorf
Sourced in Germany
The Vacuum Concentrator Plus is a lab equipment product designed to concentrate liquid samples through evaporation. It provides a controlled environment for the removal of solvents or other volatile components from samples, preserving the remaining non-volatile compounds.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Ics 5000 system, by Thermo Fisher Scientific (1 mentions)
Pda detector, by Thermo Fisher Scientific (1 mentions)
Thermo orbitrap fusion mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Thermo shaker ts 100c, by Biosan (1 mentions)
Mikro 220r, by Hettich (1 mentions)
Ethanol, by Avantor (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Pierce detergent removal spin column, by Thermo Fisher Scientific (1 mentions)
Minispin plus centrifuge, by Eppendorf (1 mentions)
Thermomixer comfort, by Eppendorf (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Millipore simplicity system, by Merck Group (1 mentions)
11 protocols using vacuum concentrator plus
1
Isolation of Insulin Lispro Derivative
2
Mass Spectrometry Analysis of PreRumA Ion
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Samples purified using ZipTips were dried using a Concentrator Plus vacuum (Eppendorf, Hamburg, Germany) and subsequently resolubilized in 50% HPLC-grade acetonitrile. Dissolved samples were directly injected into a nanoLC-coupled Thermo Orbitrap Fusion Mass Spectrometer (Thermo Fisher Scientific) with a nano electrospray ion source. The samples were introduced into a capillary which was then heated by applying a high voltage across it to produce ions that are separated and measured by the orbitrap mass analyzer. The five times charged precursor preRumA ion (m/z = 1089.72) identified in the mass spectrum (MS1) was selected and channeled to the ion-routing multipole where it was fragmented, processed in the C-trap and analyzed by the orbitrap mass analyzer (MS2). A detailed description of the MS procedure is given in a previous publication (Ongey et al., 2018 (link)).
3
Optimized Steroid Extraction from Plasma
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Steroids were extracted from mice plasma using an optimized protein precipitation protocol described by Gazárková et al. (33 ). Here, 40 µl of plasma were spiked with 2 µl of internal standards solution (50 µg/ml) and precipitated with 200 µl of acetonitrile, vortexed for 3 s, and incubated for 10 minutes at laboratory temperature. Next, the samples were centrifuged at 17,530 x g and 4°C for 10 minutes (Hettich Mikro 220R, Hettich, Germany). The supernatant was transferred to a clean Eppendorf tube and evaporated under vacuum at 60°C for 40 minutes (Vacuum Concentrator Plus, Eppendorf™, Germany). The dried residue was reconstituted prior to UHPLC-MS/MS analysis in 40 µl of 50% acetonitrile, mixed at 1400 rpm for 15 minutes at laboratory temperature (Thermo-Shaker TS-100 C, Biosan, Latvia), and transferred into the vial for LC-MS/MS analysis.
4
Extraction and Characterization of Arctium lappa Bark
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The extract of Arctium lappa was obtained from 100 g of ground and dry bark, dissolved in 1 L of 70% ethanol (JT Baker, Belo Horizonte, Brazil). A dark bottle conditioned at room temperature was used for 72 h, with daily shaking, and the extract was then filtered on filter paper. The filtrate was dried using a rotary evaporator (Eppendorf Vacuum Concentrator Plus, Hamburg, Germany). The dried extract was weighed and suspended in 997 μL PBS (phosphate-buffered saline) and 3 μL of DMSO (Dimethyl sulfoxide, Sigma-Aldrich, Saint Louis, MO, USA), until a final concentration of 1 g/mL was obtained. This extract was filtered and stored in 500 μL aliquots at −20 °C in a freezer. Plant samples were collected in the city of Uberaba, state of Minas Gerais, Brazil (Lat. −19.743623, Long −47.828514). The specimens were analyzed and identified by Prof. Dr. Milton Groppo and deposited in the herbarium of the Faculdade de Filosofia, Ciências e Letras of Ribeirão Preto, Universidade de São Paulo.
5
Mouse Genomic DNA Extraction
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400 μl tissue lysis buffer (0.5% SDS (Merck, cat. no. 112533), 100 mM NaCl, 20 mM Tris pH 8, 2.5 mM EDTA pH 8 (Merck, cat. no. 108418)) was supplemented with 80 μg proteinase K and added to each ear clip or tail cut of a mouse. The samples were lyzed for 5 to 24 h at 56°C. Afterwards, 40 μl 8 M potassium acetate and subsequently 500 μl chloroform were added to each sample and DNA was pelleted by centrifugation for 10 min/13000 rpm/RT. The resulting upper aqueous phase was carefully transferred to a new tube and mixed with 1 mL of 95% ethanol. DNA was precipitated by incubation at −80°C for 30 min and pelleted by centrifugation for 15 min/13000 rpm/4°C. The resulting supernatant was discarded, and the DNA pellets were washed once with 500 μl of 70% ethanol. The DNA was subsequently dried for 15 min at 60°C using a vacuum concentrator plus (Eppendorf). Finally, the DNA was dissolved with 125 μl water and stored at 4°C for further use.
6
Peptide Purification and Preparation for MS
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After trypsinization, peptides were eluted with ammonium bicarbonate (AmBic, 0.1 M) and acetonitrile (ACN), which were collected and dried (Vacuum Concentrator plus; Eppendorf, Germany). Dried peptides were resuspended in AmBic before DTT (5 mM) was added. Samples were incubated at 55°C for 30 min on a heat block before adding iodoacetamide (IAA, 20 mM) and incubated at RT for 30 min. DTT (5 mM) was added to stop the alkylation reaction and samples were dried. Samples were resuspended in 0.1% formic acid (FA) in H2O. Residual detergent was removed using Pierce™ detergent removal spin columns (Thermo Scientific) according to manufacturer’s protocol. The samples were then desalted with a stage tip using two filters of C18 material. The tip was first washed with methanol (MeOH) and equilibrated with 0.1% FA-H2O before samples were added. The tips were washed with 0.1% FA-H2O and samples were eluted with 0.1% FA-ACN. The samples were dried and reconstituted in 0.1% FA-H2O before mass spectrometry (MS) analysis.
7
Oligonucleotide Preparation and Quantification
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We used deionized water (Millipore Simplicity System; Millipore, Burlington, MA, USA) to prepare all aqueous solutions. To vortex the solutions, we used a Thermomixer Comfort (Eppendorf, Hamburg, Germany). Oligonucleotide solutions were concentrated on a vacuum Concentrator Plus (Eppendorf, USA). Optical density at 260 nm of oligonucleotide solutions was measured on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The oligonucleotides were precipitated and centrifuged on MiniSpin Plus centrifuges (Eppendorf, Germany).
8
Intracellular Metabolite Extraction Protocol
9
Protein Isolation and Identification
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Approximately 1 µg was loaded onto SDS–PAGE (Tris–glycine 10% polyacrylamide gel). The single protein band appeared in the gel was then excised and dried using Vacuum Concentrator Plus (Eppendorf). The proteins were extracted from dried gel pieces and digested with trypsin, and the resulting peptide sequences were subjected to analysis (MALDI-TOF MS/MS—Proteomics International Laboratories Ltd, Australia).
10
Extraction and Preparation of L. inflata Bark
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The extract of L. inflata was obtained from 100 g of ground and dry bark, dissolved in 1 L of 70% ethanol (JT Baker, Belo Horizonte, Brazil). A dark bottle conditioned at room temperature was used for 72 hours with daily shaking, and the extract was then filtered on filter paper. The filtrate was dried using a rotary evaporator (Eppendorf Vacuum Concentrator Plus, Hamburg, Germany).
The dried extract was weighed and suspended in 997 μL PBS (phosphate-buffered saline) and 3 μL of DMSO (Dimethyl sulfoxide, Sigma-Aldrich, Missouri, USA) until a final concentration of 1 g/ mL was obtained. This extract was filtered and stored in 500 μL
The dried extract was weighed and suspended in 997 μL PBS (phosphate-buffered saline) and 3 μL of DMSO (Dimethyl sulfoxide, Sigma-Aldrich, Missouri, USA) until a final concentration of 1 g/ mL was obtained. This extract was filtered and stored in 500 μL
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