Beckman p ace mdq system

The separation and detection of NP proteins and NP aptamers mixtures were carried out using a Beckman P/ACE MDQ system (Beckman-Coulter, Fullerton, CA, USA) equipped with a LIF (Laser-Induced Fluorescence) detector, enabling the visualization of FAM-labeled NP aptamers. Analysis of all CE data was performed using 32 Karat software. Separation was achieved using an uncoated fused silica capillary (75 μm i.d. × 50.2 cm (40.0 cm effective), Sino Sumtech, Handan, Hebei, China) at a constant temperature of 25 °C. Sample injection into the capillary was conducted at a pressure of 0.5 psi for 10 s, and separation was driven by a running voltage of 20 kV (498 V/cm). Excitation was generated using the 488 nm line of an Ar+ laser (Beckman Coulter), with emission collected at 520 nm. The running buffer consisted of 50 mM H₃BO₃/Na₂B₄O₇ (pH 7.8). To mitigate potential adsorption of NP proteins on the capillary’s inner surface and ensure reproducible CE separation, the capillaries underwent a treatment sequence involving 0.1 M NaOH for 3 min, followed by water for 3 min, and running buffer for an additional 3 min after each consecutive sample injection.

Intracellular sodium content (Na⁺i) was measured by capillary electrophoresis [41 (link),42 (link)]. Briefly, rbcs (3 10⁶ cells) were attached to 0.001% poly-D-lysine-coated coverslips and exposed to isosmotic medium (basal) or MST7 10 μM for 1, 3 or 5 min. To remove extracellular sodium, cells were washed three times with medium containing Hepes 20 mM, imidazole 15 mM and sucrose, 300 mosM, pH 7.4. LiCO₃ was added to the assay as internal standard (3 μL, 4 mM) and cells were then lysed by exposure to TCA (4% at 4°C for 30 min). The lysate was centrifuged (18000 x g at 20°C for 5 min) and the resulting supernatant was dried in a vacuum concentrator for 2 hours (Savant^™ SPD131DDA SpeedVac^™ Concentrator). The samples were then reconstituted in ultrapure water and stored at 4°C.
Capillary electrophoresis was performed with a Beckman P/ACE MDQ system (Beckman Coulter, Brea, CA, U.S.A.), equipped with a diode array detector. Fused-silica capillaries were 60cm in total length (50 cm from the inlet to the detector) and 75 μm id. The running background electrolyte consisted of Imidazol 5 mM and HIBA 6.5 mM, pH 3.0. Runs were carried out in normal mode (cathode at the outlet side, 20 kV) and detection was indirect at 254 nm.
Results were expressed as relative intracellular sodium content, where sodium basal value is 1 and sodium changes are expressed as relative to the initial value.