SGC3

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).
1. Stock Solution Preparation
1.1 Solvent: HEPES buffer [50 mM HEPES and 100 mM NaCl (pH 7.4)].
1.2 Concentration recommendation: 30 μM.

3. Staining Procedure
3.1 Sample type: 30% PE/70% PC liposomes.[1]
3.2 Incubation conditions:
3.2.1 For melittin pores: Incubate dye-loaded liposomes with melittin at melittin:lipid molar ratios of 1:10, 1:20, or 1:800 for 5 min at room temperature.
3.2.2 For αHL-wt or αHL-M113W pores: Incubate dye-loaded liposomes with toxin at αHL:lipid molar ratios of 1:14, 1:50, or 1:300 for 1 h at 37°C.
3.3 Washing steps: Rapidly dilute liposome-toxin mixtures in HEPES buffer [50 mM HEPES and 100 mM NaCl (pH 7.4)].

4. Controls
4.1 Set up blank dilution controls (liposomes diluted in the absence of pore-forming toxin).

5. Detection & Analysis
5.1 Instrument: Spectrophotometer or stopped-flow spectrometer.
5.2 Result analysis:
5.2.1 Fluorescence intensity decreases over time as dye effluxes from liposomes.
5.2.2 Time constant for efflux through melittin pores is 8.3 ± 0.3 s; through αHL-wt pores is 8.0 ± 0.4 s; through αHL-M113W pores is 29.3 ± 3.1 s.