P 1000 flaming brown micropipette puller

Current-clamp recordings of alpha cell membrane potential were performed using the perforated-patch configuration. Patch electrodes were fabricated from borosilicate glass tubes using a P-1000 Flaming/Brown micropipette puller (Sutter Instrument, Novato, CA) and had a tip resistance of 3–6 MΩ when filled with pipette solution. The pipette solution contained (in mM): K₂SO₄ 76, KCl 10, NaCl 10, MgCl₂ 1, HEPES 5 (pH 7.3 with KOH), and 0.15 mg/ml amphotericin B. The bath solution contained (in mM): NaCl 140, KCl 3.6, MgSO₄ 0.5, CaCl₂ 1.5, HEPES 10, NaH₂PO₄ 0.5, NaHCO₃ 5, glucose 15 (pH 7.4 with NaOH). Recordings were acquired using Axopatch 200B amplifier, Axon Digidata 1550B, and pClamp 10 software (Molecular Devices, San Jose, CA) at 10 kHz and filtered at 1 kHz.

The CZE system consists of two high-voltage power supplies (Spellman CZE 1000R) and an electrokinetically pumped electrospray interface.^{3 (link),28 (link)} The interface was used to couple the CZE separation capillary to a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). The electrospray emitter was made from borosilicate glass capillary (1.0 mm o.d. × 0.75 mm i.d., 10 cm long) pulled with a Sutter instrument P-1000 flaming/brown micropipette puller; the size of the emitter opening was 5–10 μm. The electrospray sheath flow buffer was 10% (v/v) methanol with 0.1%FA. A commercial LPA coated capillary (50 μm i.d.×150 μm o.d., 60 cm long) was used for the CZE separation. The separation buffer was 50 mM FA in water. A 20 nL injection volume was loaded by pressure. 19.5 kV was applied at the injection end of the capillary for separation and 1.5 kV on the sheath flow reservoir for electrospray. Voltage programming was controlled by LabView software.

An ECE-001 CE autosampler and a commercialized electro-kinetically pumped sheath flow CEMS interface from CMP Scientific (Brooklyn, NY) was used [28 –30 (link)]. A fused silica capillary (1-m-long, 50 μm i. d., 360 μm o. d.) was coated with linear polyacrylamide (LPA) according to references [31 ] and [32 (link)]. The outer diameter of one end of the capillary was reduced to ~70–80 μm by etching with hydrofluoric acid based on reference [28 ]. (Caution: use appropriate safety procedures while handling hydrofluoric acid solutions.)
Samples were injected into the capillary by applying pressure for a specified amount of time to achieve the necessary volume and sample amount based on Poiseuille’s law. Loading volume was 200 nL for the 40-ng standard protein analysis and 500 nL for the 1 μg E. coli protein analysis. A separation voltage of 30 kV and 20 kV was used for the standard protein sample and E. coli sample, respectively. The capillary was flushed between runs with background electrolyte (BGE) with a pressure of 10 psi for 10 min. A glass capillary (1.0-mm o. d., 0.75-mm i. d., 10 cm long) was pulled with a Sutter P-1000 flaming/brown micropipette puller and used as the ESI emitter. Emitter orifice was 20–40 μm with a voltage for ESI of about 2 kV. BGE for CZE was 5% (v/v) acetic acid (pH 2.4) and the sheath buffer consisted of 0.2% (v/v) formic acid and 10% (v/v) methanol.