Valinomycin was extracted with three-fold volumes of ethyl acetate from cell-free reactions. After centrifugation at 16,000 g for 5 min, the organic fraction was transferred to a fresh 1.5 mL microcentrifuge tube, air dried, and resuspended in methanol for Valinomycin analysis. Valinomycin quantification was performed with an Agilent 6470 Triple Quadrupole LC/MS System equipped with an Eclipse Plus C18 column (2.1×50 mm, 1.8 μm). For Valinomycin detection, 2 μL of each sample was injected and separated at a flow rate of 0.4 mL/min with elution buffers A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid) through a linear gradient elution from 80 to 100% B over 2.5 min, a 100% B wash for 7.5 min, and a post time wash for 10 min with 80% B. Valinomycin concentrations were calculated according to a calibration curve prepared with commercial Valinomycin (Sigma) as a standard. All measurements were performed in triplicate.
Valinomycin
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe, Italy, Switzerland, United Kingdom, Spain
Valinomycin is a macrocyclic compound produced by Streptomyces bacteria. It functions as an ionophore, facilitating the transport of potassium ions across cell membranes.
Automatically generated - may contain errors
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143 protocols using valinomycin
1
Quantification of Valinomycin by LC-MS
2
Transmembrane Potential Gradient in Lipid Vesicles
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In order to create a concentration gradient between the inside and outside of the vesicles, the non-encapsulated potassium was removed by filtration: lipid vesicles (1 mL) were loaded on a 20 cm Sephadex G-50 coarse (Pharmacia, Uppsala, Sweden) filtration column, pre-equilibrated with 5 μM glycine (pH 7.0 ± 0.5) containing an appropriate concentration of sucrose to make the osmotic pressure of the equilibration solution equal to that of the internal vesicle, thus avoiding vesicle disruption during separation. The vesicle-containing fraction was collected by measuring turbidity at 403 nm41 (link), and immediately used for subsequent experiments.
In order to create a transmembrane potential, experiments with valinomycin added were done in the following steps. The vesicle-containing fraction (1.2 mL) was diluted in 3 mL of an equi-osmolar sucrose solution (5 μM glycine-200 mM sucrose, pH 7.0 ± 0.5) and poured into the axon model, then 5 µL of valinomycin (10 mg/ml, Sigma-Aldrich) was added to a final concentration of 15 mM. The approximate molar ratio of valinomycin to lipid was 2.9 × 10−3.
In order to create a transmembrane potential, experiments with valinomycin added were done in the following steps. The vesicle-containing fraction (1.2 mL) was diluted in 3 mL of an equi-osmolar sucrose solution (5 μM glycine-200 mM sucrose, pH 7.0 ± 0.5) and poured into the axon model, then 5 µL of valinomycin (10 mg/ml, Sigma-Aldrich) was added to a final concentration of 15 mM. The approximate molar ratio of valinomycin to lipid was 2.9 × 10−3.
3
Mitochondrial Dysfunction in LRRK2 PD Fibroblasts
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Previously described (Smith et al., 2016 (link), Korecka & Thomas et al., 2019) deidentified healthy and LRRK2 G2019S patient fibroblasts were obtained from the Coriell and the NIA Aging Cell biorepositories or the Mayo clinic. Conducting research with patient fibroblasts was not considered to be human subject research as it does not involve intervention or interaction with the individual and the samples do not contain identifiable private information. The average age of the control and the mutant fibroblasts was matched to 66 and 67 years, respectively. Mitochondrial super oxide (MitoSOX™ Red, ThermoFisher Scientific, M36008) levels, induced by 24-hour treatment with the mitochondrial depolarization agent valinomycin (10μM, SigmaAldrich, V0627), were measured in human fibroblasts obtained from healthy individuals (N=4) and PD patients carrying a LRRK2 G2019S mutation (N=5), in the presence of sepiapterin or SPRi3 (pretreatment for 24-hours before addition of valinomycin), using the live cell imaging system IncuCyte.
4
Dissecting the Role of ΔΨ and ΔpH in Ipa Protein Export
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To test the role of the Δ Ψ and Δ pH in Ipa proteins export, we used carbonyl cyanide m-chlorophenyl hydrazone (CCCP, Sigma) as previously described [29 (link), 31 (link)]. Briefly, cells at mid-log growth stage were washed twice in TCSB medium (pH ~7.3) containing 120 mM Tris (pH 7.0), and resuspended in TCSB containing either 0.5% DMSO or CCCP at concentrations up to 40 μM at 37°C for 30 min. To test the role of the ΔΨ alone, we used valinomycin (Sigma) as previously described [29 (link), 31 (link)]: cells were washed further in TCSB containing 150 mM KCl after Tris treatment, then resuspended in TCSB containing 150 mM KCl supplemented with either 0.5% DMSO or valinomycin at concentrations up to 40 μM at 37°C for 30 min. To test the role of the Δ pH alone, cells were washed twice in 200 mM sodium phosphate buffer, pH 5.85, then resuspended in the same buffer containing potassium benzoate (Sigma) [56 (link)] at concentrations up to 40 mM at 37°C for 30 min. Both cells and supernatants were collected and analyzed by immunoblotting using the antibodies indicated in the figures.
5
Isolation and Characterization of B. cereus
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B. cereus CAU45 was isolated from a raw milk sample in one local dairy farm of Beijing, China. B. cereus DSMZ 4312 was used as a reference strain for cereulide and Nhe, while B. cereus ATCC 14579 was chosen as a reference strain for Nhe and Hbl. All the monoclonal antibodies used for the detection of NheA and NheB, and CBP were as previously described [27 (link)]. HEp-2 cells used to evaluate the cytotoxicity of cereulide were obtained from Prof. Xiaojia Wang at China Agricultural University. Valinomycin purchased from Sigma-Aldrich (St. Louis, MO, USA) was used as an internal standard by UPLC-MS/MS test.
6
Peristaltic Pump-Driven Biosensor Fabrication
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CNT/isopropanol slurry (TNAPM-M8) was purchased from Chengdu Organic Chemicals Co. Ltd. GO aqueous solution (Carmery) was purchased from the Institute of Coal Chemistry, Chinese Academy of Science. Ag/AgCl conductive inks were purchased from ALS Co. Ltd. All of the following chemicals were obtained from Sigma-Aldrich: BSA, glutaraldehyde solution, selectophore grade sodium ionophore X, sodium tetrakis [3,5-bis(trifluoromethyl)phenyl] borate (Na-TFPB), valinomycin (potassium ionophore), sodium tetraphenylborate (NaTPB), bis(2-ethylehexyl) sebacate (DOS), high–molecular weight polyvinyl chloride (PVC), tetrahydrofuran, cyclohexanone, dimethyl sulfoxide, polyaniline emeraldine, sodium chloride (NaCl), and Gox (from Aspergillus niger). All the chemicals were used as received. All solutions were prepared using deionized water (18.3 megohm·cm) produced from a Millipore water purification system, unless otherwise noted. The peristaltic pump was purchased from Aquatech Co. Ltd. (Osaka, Japan), which mainly consists of a motor, a pump head, and a tube. The tube is fixed between the pump head and the pump case. The motor drives the pump head to rotate, which alternately squeezes and releases the tube to pump fluid. As the pump head rotates, negative pressure forms inside the tube, which allows the liquid to flow through the tube.
7
Electrochemical Sensor for Human Serum
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High-molecular-weight PVC (HMW
PVC), potassium ionophore I (Valinomycin), bis(2-ethylhexyl) sebacate
(DOS), tetrahydrofuran (THF) (all selectophore grade), anhydrous acetonitrile
(ACN, 99.5%), anhydrous dimethylformamide (DMF, 99.8%), sulfuric acid
(H2SO4, ACS reagent, 95–98%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (EDC, 98%), N-hydroxysuccinimide (NHS),
4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-amino-TEMPO, free
radical), silver nitrate (AgNO3), and tetraethylammonium
nitrate (TEANO3) were purchased from Sigma-Aldrich. Tetrabutylammonium
hexafluorophosphate (TBAPF6, for electrochemical analysis,
≥99%) and nitric acid (≥69%) were obtained from Fluka.
Potassium tetrakis(pentafluorophenyl)borate (KTFAB, 97%) was received
from Alfa Aesar. Multiwalled carbon nanotubes (MWCNT) were purchased
from HeJi, Inc. (30–50 nm diameter, 0.5–200 μm
length, M4905). Human serum was received from HyTest Ltd. (Lot. 15/05-8TFS)
and Merck Millipore (Lot. 3733758). Absolute ethanol and methanol
(AR grade) were obtained from Molar Chemicals Ltd. (Halásztelek,
Hungary). The aqueous solutions were prepared with deionized water
(DIW) with a resistivity of 18.2 MΩ cm.
PVC), potassium ionophore I (Valinomycin), bis(2-ethylhexyl) sebacate
(DOS), tetrahydrofuran (THF) (all selectophore grade), anhydrous acetonitrile
(ACN, 99.5%), anhydrous dimethylformamide (DMF, 99.8%), sulfuric acid
(H2SO4, ACS reagent, 95–98%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (EDC, 98%), N-hydroxysuccinimide (NHS),
4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-amino-TEMPO, free
radical), silver nitrate (AgNO3), and tetraethylammonium
nitrate (TEANO3) were purchased from Sigma-Aldrich. Tetrabutylammonium
hexafluorophosphate (TBAPF6, for electrochemical analysis,
≥99%) and nitric acid (≥69%) were obtained from Fluka.
Potassium tetrakis(pentafluorophenyl)borate (KTFAB, 97%) was received
from Alfa Aesar. Multiwalled carbon nanotubes (MWCNT) were purchased
from HeJi, Inc. (30–50 nm diameter, 0.5–200 μm
length, M4905). Human serum was received from HyTest Ltd. (Lot. 15/05-8TFS)
and Merck Millipore (Lot. 3733758). Absolute ethanol and methanol
(AR grade) were obtained from Molar Chemicals Ltd. (Halásztelek,
Hungary). The aqueous solutions were prepared with deionized water
(DIW) with a resistivity of 18.2 MΩ cm.
8
Measuring Intracellular pH in HEK293 Cells
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pHi of HEK293 cells was measured in cells grown on coverslips coated with PEI. The coverslips were placed in a custom designed chamber on the stage of a microscope fluorometer and loaded with the fluorescent pHi probe BCECF using esterified BCECF‐AM (Life Technologies) at room temperature for 20 min. The composition of the solution used for dye loading and each of the various buffered solutions used is shown in Table 1 . In each experiment, the fluorescence excitation ratio (500/440 nm; 530‐nm emission) obtained from 200 cells was averaged. The bathing solutions continuously perfused the coverslips at 2 ml/min (37°C). At the end of each experiment, the intracellular fluorescence excitation ratio was calibrated using 5 mM valinomycin (Sigma‐Aldrich) and 26 mM nigericin (Sigma‐Aldrich). In all groups as in the lysosomal measurement experiments, the cells were bathed in the Hepes‐buffered solution followed by either bicarbonate‐buffered, bicarbonate/carbonate (carbicarb)‐buffered or THAM‐buffered solutions.
9
Characterization of P-gp Inhibitors
10
Mitochondrial Respiration Assay
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Cyclosporin A (CsA), rotenone, rhodamine 123 (Rh123), dithiothreitol (DTT), monobromobimane+ (MBM+), hematoporphrin (HP), oligomycin, valinomycin and MOPS were purchased from Sigma (St. Louis, MO). All other reagents were of analytical reagent grade, and all solutions were prepared with asepsis double-distilled water.
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