Table 1.
Comparison of assay and hardware related parameters in SSME recordings on TMEM175 using the SURFE2R N1 and the SURFE2R 96SE.
| Parameter | SURFE2R N1 | SURFE2R 96SE |
|---|---|---|
| Assay parameter | ||
| Main buffer (M) 1 | 30 mM HEPES, 30 mM MES, 5 mM MgCl2, 90 mM NaCl, pH 7.6 (NaOH) | |
| Non-activating solution (NA) 1 | M + 50 mM NaCl | |
| Activating solution (A) 1 | M + 50 mM KCl | |
| Total protein concentration of the sample | 2.2 mg/mL | |
| Sample dilution used per sensor well | 1:10 | 1:100 |
| Sample volume per sensor | 10 µL | 8 µL |
| Sample consumption based on total protein concentration determined via Bradford assay | 2.2 µg protein per sensor | 0.18 µg protein per sensor; 17 µg protein per plate |
| Typical mode of measurement | Sequential recordings on the same sensor using different buffer compositions (i.e., pH or concentration sequence); first, three measurements using standard conditions; in-well normalization of dataset to standard activation response before averaging across sensors | Parallel recordings on different sensors; first, three measurements using standard conditions; then, one assay condition is applied per sensor (i.e., pH, ion concentration, compound addition); in-well normalization of single data point to standard activation response before averaging across sensors |
| Hardware parameter | ||
| Parallelization | Single well | 96 wells |
| Sensor diameter | 3 mm | 3 mm |
| Liquid handling of solution exchange | Continuous solution flow: 1 s NA, 1 s A, 1 s NA |
Stack of solutions in 200 µL pipette: 50 µL NA 2, 30 µL A, 80 µL NA |
| Restoring initial conditions | Rinse with 1 mL NA at continuous flow conditions | Stepwise dilution: remove solution down to ~30 µL well volume; rinse with 200 µL NA; remove solution down to ~30 µL well volume; refill with 60 µL NA |
| Solution flow speed (defines time resolution) | 200 µL/s | 200 µL/s |
| Time resolution taken from [33] | 38 ms | 7 ms |
| Measurement time window (time of A flow) | 1 s | ~250 ms |
| A consumption per measurement | 300 µL (200 µL + 100 µL spare) | 50 µL |
| NA consumption per measurement | 1.6 mL (Measurement: 600 µL; rinse: 1 mL) |
~0.4 mL (Measurement: 130 µL; rinse + refill: 260 µL) |
| Solution consumption per compound and well in compound assay | ~2 mL (rinse, incubate, measure) | ~0.4 mL (rinse, incubate, measure) |
| Read-out characteristics | ||
| TMEM175 peak current | (12 ± 3) nA | (4.1 ± 1) nA |
| Control peak current | (−1.07 ± 0.3) nA | (−1.29 ± 0.52) nA |
| TMEM175 rise time constant τ1 | (5.1 ± 1.3) ms | (9.2 ± 1.7) ms |
| TMEM175 decay time constant τ2 | (10.7 ± 1.1) ms | (10.2 ± 0.8) ms |
| TMEM175 system time constant τ3 3 | (285 ± 71) ms | N.A. |
1 This buffer composition represents our standard condition, which is used to examine the modulation of K+ flux through TMEM175, i.e., in our compound assays. 2 The first NA represents the initial solution, which is taken from the sensor well instead of the reservoir. This compensates for slight differences of the well solution compared to NA solution in the reservoir and prevents solution exchange artifacts. 3 The system time constant is a consequence of capacitor discharging and affected by capacitance and conductance of the compound membrane on the sensor [32]. It is only visible for fast currents in SSME and does not provide information about TMEM175, but rather represents properties of the sensor.