Reconstitution of Functional Integrin into Phospholipid Vesicles and Planar Lipid Bilayers - Methods in Molecular Biology: Extracellular Matrix Protocols - page 72

Fig. 5. Association and dissociation kinetics of peptide binding to α IIb β 3 in bilay-

ers by TIRFM. Bilayers with 3 in DMPG/DMPC vesicles were formed by vesicle

fusion. TRITC-labeled peptide GRGDSPC was added at a concentration of 3300 n M
and association kinetics were followed by measuring the time course of the fluores-
cence intensity (

α IIb β

) at an excitation of 514 nm. After reaching the equilibrium, the cell
was washed with Buffer C (4 mL/min) while monitoring the time-course of fluores-
cence intensity. TRITC-labeled GRGDSPC-peptide could be dissociated by addition
of a 100-fold molar excess of unlabeled GRGDS-peptide. The background signal of
about 180 relative fluorescence units was caused by labeled peptide which stayed free
in solution and could be abolished by washing with buffer.

°

2.2. Vesicle Preparation
1. Lipid stock solution: lyophilized phospholipids DMPG and DMPC (Avanti Polar

Lipids, Alabaster, AL) were dissolved in chloroform/methanol 2:1 at a concen-
tration of about 15 m M . Precooled solvent (30 min at -20 ° C) is easier to pipet

( see Note 2 ).
2. 2 M (w/v) Tris-HCl, adjust pH to 7.4 with concentrated HCl.
3. 5 M (w/v) NaCl, warm the solution to facilitate dissolvation.
4. Buffer A: 20 m M Tris-HCl pH 7.4, 50 m M NaCl, 1 m M CaCl 2 .
5. Buffer B: Dissolve 45.4 L Triton X-100 in buffer A, final volume 50 mL, final

concentration 0.1% (w/v) ( see Note 3 ).

µ

6. About 500 mg of BIO-Beads SM-2 (Bio-Rad, Richmond, CA) were first washed

with 50 mL methanol and then with 500 mL distilled water. The wet beads were
collected on a sintered glass funnel ( see Note 4 ).