Working With Lipids And Membranes In BIACORE
(Standard Operating Procedure)
Preparation of Lipid Vesicles
(See also "Preparation of Liposomes" from Avanti Polar Lipids, Inc.)
1) Dissolve phospholipids in ethanol free chloroform to ~10mg/ml.
2) Add lipids at desired composition to glass corex tubes or round bottom flask and deposit as a thin film by removal of the solvent under reduced pressure on a rotary evaporator, then dry under high vacuum for 2h.
A Lipids must be hydrated at temperatures above their phase transition temperature
(Tm).
3) Gently resuspend lipid in phosphate buffer, pH 7.4 to give a final concentration of 20mM. During mixing it is important not to generate bubbles.
4) Shake lipid suspension for 30 min and then sonicate in a bath sonicator for 2 min.
A Hydration time may differ slightly among lipid species and structure, however, a hydration time of 1 hour with vigorous shaking, mixing, or stirring is highly recommended.
5) To obtain small unilamellar vesicles, pass lipid suspension ~17-21 times through a 50nM polycarbonate filter using extrusion apparatus to give a translucent solution.
6) Sonicate the lipid suspension on ice under a stream of nitrogen using a direct probe,
3 x 10 min at 6m amplitude (or low setting). A bath sonicator may also be used.
A Ultracentrifugation is important for obtaining a homogenous lipid preparation.
7) The lipid suspension is then purified by ultracentrifugation. Centrifuge the lipid sample for 25 min at 72000 rpm at 15°C with TL-100, (or for 30 min at 35000 rpm with the Beckman rotor SW55Ti on Beckman ultracentrifuge, followed by a 40,000 rpm spin for 3 hours to remove large multilamellar vesicles and metal particles.
8) The purified lipid will be in the upper layer of the suspension. Transfer the lipid layer using a Pasteur pipet to a new tube and store at 4°C under N2.
Preparation of Membrane Extracts
1) Harvest cells with standard EDTA or Trypsin solution. EDTA is preferred because no additional protein is introduced. For cells in suspension, harvest by spinning cells down at 5000xg for 20 min.
2) Resuspend cells in PBS or Tris buffer containing appropriate stabilizers and centrifuge at 5000xg for 20 min.
Note: pellets can be frozen at this point if necessary.
3) Resuspend pellet in a hypotonic lysis buffer (i.e. 10mM Tris/HCl pH 7.4, 0.5mM PMSF, and 0.01 mg/ml aprotinin). Use 4 mls per ml of pooled cells.
4) Rupture cells by freeze thawing 4-5 times with dry ice/ ethanol bath and a 37-42°C water bath. Homogenization may also be used.
5) Spin homogenate at 7000xg for 15 min to remove unbroken cells and large debris.
6) Pellet membranes by spinning supernatant at 200,000xg for 90 min.
7) If working with native membranes then resuspend pellet in buffer containing 0.5mM PMSF and 0.01 mg/ml aprotinin.
8) If detergent solubilization of membranes is necessary, proceed by resuspending in appropriate detergent and proceed with membrane solubilization protocol.
A Procedures that employ detergents vary depending upon the type of detergent; however, the principle is the same. Lipids are solubilized by the detergent of choice (such as cholate or octylglucoside); then the detergent is removed either rapidly by dilution or gel filteration, or slowly by dialysis. As the detergent concentration decreases, the lipids adopt unilamellar vesicular structures.
9) A final high speed spin (greater than 100,000xg) of the membrane prep supernatant will pellet the microsomal fraction.
10) Resuspend membrane pellet in buffer (may need to homogenize to resuspend completely), then do a protein determination, aliquot membrane suspensions and store at &endash;80°C.
Formation of Lipid Layer on the Sensor Surface
A Lipid layers are best formed at temperatures above the phase transition temperature of the lipid used. At 25°C, egg yolk PC, DMPC, and unsaturated lipids such as POPC should result in a fluid mono- or bilayer.
A Make sure instrument is clean and detergent free. Running buffers should be filtered and rigorously degassed daily. The following steps can be used for both the HPA and L1 sensor chip.
1) Clean sensor chip with 100ml of 40mM octyl D-glucoside at a flow rate 10ml/min.
2) Clean injection needle by pre-dipping in water.
3) Immediately inject SUV (20ml, 500mM) or membrane fractions at a flowrate of 2ml/min. (If working with membranes, sonicate just prior to injection to break up any membrane fragments).
4) Wash lipid layer at 100ml/min with NaOH (20mM, 20ml) and condition surface with regeneration buffer (if regenerations conditions are known). (Do not use NaOH if working with natural lipids)
5) Inject BSA (0.1 mg/ml) for 5 min to block non-specific binding (should bind ~100 RU).
What To Expect
* Stable signal of ~2000 RU on HPA and ~3000 RU on L1 after the NaOH wash.
* Lipid membrane should be stable to short exposures of high salt, acid, and base (i.e.
2M NaCl, 100mM HCl, 100mM NaOH)
* Degree of non-specific binding of BSA will increase with repeated cycles of lipid loading and cleaning with detergent.
Control Surfaces
A As a general rule, always place control surface in flow cell 1 to prevent contamination from other flow cells.
® For HPA chip inject BSA (0.1 mg/ml) for 5 minutes over blank flow cell. Surface will saturate at ~1000 RU.
® Lipid alone (without protein) can be injected over a blank surface to be used as a
control. PC vesicles can also be used on a control surface.
® For the L1 chip, lipid alone (without protein) can be injected over a blank flow cell, or PC vesicles can also be used as a control surface.
Deposition of Ligands
l Small acylated ligands (<1000 Da) can be inserted directly into a lipid monolayer by
injection of dilute solutions (~50 mM) across the monolayer at a flow rate of 10ml/min.
l Larger molecules tend not to associate with the pre-formed lipid layer. Deposition can be achieved by shaking purified protein (~100nM) for 5 minutes with vesicles formed by extrusion (~500mM) in phosphate buffer followed by loading the surface with the protein containing vesicles.
Regeneration and Stability of the Surface
t Analytes may be dissociated with variations in pH and ionic strength, leaving the
lipid layer intact for additional cycles of interaction.
t Suggested reagents that will not destabilize most lipid layers, injected for 2 minutes at
a flow rate of 20ml/min:
DMSO, 10% Ethanolamine, 10%
Ethanol, 10% NaOH, 100mM
HCl, 100mM NaCO3, 10mM
Glycine, pH2, 10mM KCl, 2M
NaCl, 2M
t Detergents and organic solvents will alter or destablize the membrane or liposomes.
CHAPS or octyl D-glucoside may be used to strip the lipids from the surface in preparation for binding new membranes or liposomes.
A Natural lipids should not be exposed to NaOH due to possible modification of
the head groups.