O-Labeling Experiments: O-Enriched water (90 μL, O content 97 %) was added to a buffer solution (OH2 10 μL, 1 M Tris/HCl pH 8.0) to reach a final buffer concentration of 100 m M (18/16O-label 18/16O-label
The pellet was washed with buffer, recentrifuged for 1 h, and resuspended in buffer to reach a lipid concentration of 10 mg/mL; 1 2 μL of proteoliposome suspension was injected into the cis compartment, followed by membrane rupture and formation of a new membrane.
IMS was introduced in the 1970s [ 67] and utilizes the fact that ions subject to an electric field in a buffer gas quickly reach a steady velocity dependent on the ion shape: compact species drift faster than those with extended structures [ 68, 69].
The mixture was transferred to a 15 mL centrifuge tube with isolation buffer to reach a final volume of 3 mL.
For some experiments, additional NaHCO3 was added along with the borate buffer to reach a bicarbonate concentration between 0.1 and 10 mM.
After approximately 30 min, the resonance frequency of the pure liposome channel reached a minimum and then increased again owing to the release of trapped aqueous buffer to reach a stable value of Δ ffinal=−27±3 Hz (Table 1).
Treatments were conducted by pipetting 10 µL ABA solution or the respective amount of EtOH as solvent control diluted in assay buffer to reach a final concentration of 10 50 µM ABA and 0.01 0.05 % EtOH.
Trypsin was dissolved in 10 μl of buffer to reach a trypsin-to-protein mass ratio of 1 50 in the final solution; the trypsin solution was added to the sample, which was incubated at 37 °C overnight for 15 h.
To perform the coimmunoprecipitation, 50 μg of total protein was diluted in coupling buffer to reach a final volume of 400 μL, and this mixture was added to the corresponding columns, which were incubated for 8 hr at 4°C using an orbital mixer.
Phosphatidylcholine, cholesterol, and the essential oil of Atractylodes macrocephala Koidz were dissolved in a mixture of scCO2/ethanol, and after the system reached equilibrium, a buffer solution was injected by a syringe pump into the dissolved solutes.
Preliminary experiments using this preparation, and based on direct NADP/NADPH-based fluorimetric measurements (20), indicate that at physiological pH 7.4, the maximal obtainable glutamate-filling of the pure vesicles is reached using a buffer containing 50 mM glutamate.
