Real time interaction between immobilized liposomes and lipid binding proteins by Surface Plasmon Resonance

From LipidomicsWiki

Real time interaction between immobilized liposomes and lipid binding proteins by Surface Plasmon Resonance

Equipment
- Biacore ® 3000 instrument (GE Healthcare)
- Pioneer® L1 sensor chip (GE Healthcare)

1. Preparation of liposomes (large unilamellar vesicles)
- mix appropriate quantities of lipid stock solutions (in organic solvents)
- evaporate to dryness under nitrogen
- resuspend in appropriate buffer (e.g. PBS; may differ from running buffer) to a lipid concentration of 0.5 mM
- freeze/ thaw six times
- sonicate for 2 x 30 sec
- pass 19 times through a polycarbonate membrane (pore diameter 100 nm, Avanti Polar Lipids) in a mini-extruder (Liposofast ®, Avestin)
- optional: quantify lipid content of an aliquot by standard TLC method, determine liposomal size by dynamic light scattering

2. Immobilization of liposomes
Load the L1 chip with liposomes until saturation is reached (about 4000-10,000 response units (RP), depending on liposomal composition). Loosely associated liposomes or multilamellar structures are removed by NaOH.

- inject 60 µl liposome suspension at a flow rate of 5 µl/min
- inject 10 µL NaOH (25 mM) at 5 µl/min
- inject 20 µl liposome suspension at 5 µl/min
- inject 10 µL NaOH (50 mM) at 100 µl/min
- inject running buffer until baseline is stable. Composition of running buffer is optional and should physiologically relevant conditions.

3. Interaction measurement
Injection volume/ flow rate/ protein concentration and temperature may be varied.
Experiments should at least be performed in duplicates, taking into consideration the gradual degeneration of the chip surface.
Quantitative evaluation – if possible (i.e. no disintegration of the liposomal surface and lipid loss occur) - is performed using the protocols of the Biacore software or algorithms of one’s own.

- association phase: inject 60 µl protein in running buffer (several mM) at 20 µl/min (= 3 min)
- dissociation phase: inject 60 µl pure running buffer at 20µl/min (= 3 min)

4. Regeneration of the chip
- injection of 40 µl CHAPS (20 mM) at 20 µl/min
- injection of 10 µl NaOH (50 mM) at 100 µl/min
- if the original baseline is not reached after this procedure, use SDS and /or organic solvents according to manufacturer’s instructions

5. Source
modified from: <ref>Remmel, N., et al., Saposin B mobilizes lipids from cholesterol-poor and bis(monoacylglycero)phosphate-rich membranes at acidic pH. Unglycosylated patient variant saposin B lacks lipid-extraction capacity. FEBS J, 2007, 274, 3405-20.</ref>