Reconstitution of Functional Integrin into Phospholipid Vesicles and Planar Lipid Bilayers - Methods in Molecular Biology: Extracellular Matrix Protocols - page 74

3. Methods
3.1. Fluorescence Labeling of Proteins
1. Integrin α IIb β 3, dissolved in buffer B, is adjusted to pH 8.0 by addition of 0.2 M

NaHCO 3.

2. Add a 50-fold molar excess of FITC or TRITC to the protein, vortex shortly.

3. Incubate the mixture for 30 min in the dark at room temperature.

4. Remove excess of FITC and TRITC by gelfiltration on a Sephadex G-25 M col-

umn, equilibrated with Buffer B.

5. Measure the OD of the FITC- or TRITC-labeled protein at 493 nm

( = 151.5 cm 2 /mg), respectively. The degree of

labeling can be calculated according to the following equation: c = OD/

ε = 153.8 cm 2 /mg) or 555 nm ( ε

*M* d .
The degree of labeling is between 1-2 FITC or TRITC groups per integrin.

ε

3.2. Vesicle Preparation
1. Take an aliquot corresponding to 435 nmol of each lipid solution using
glass micropipets.
2. Dry lipids first under an N 2 -stream, then in high vacuum for 2 h.
3. Add 930 L Buffer B to the lipid film and vortex the solution until the lipid film

is completely solubilized. Transfer to a 1.5-mL Eppendorf tube.

µ

4. Add 70 µ L integrin α IIb β 3 dissolved in Buffer B (3 mg/mL) to the lipid solution.

5. Shake the mixture for 120 min at 37 ° C.

6. Add 50 mg of washed BIO-Beads and shake for 210 min ( see Note 6 ).

7. Spin briefly to precipitate the beads and transfer the supernatant into a new tube.

Add a second 50 mg of BIO-beads ( see Note 7 ).

8. Spin again, remove the supernatant, it should now be opalescent. Load the super-

natant on top of the sucrose gradient and fill the tube with Buffer A.

9. Use a TST60 (Beckman, Fullerton, CA) or equivalent vertical rotor and centri-

fuge at 4 ° C and 275,000 g for 24 h.

10. The vesicle-containing band can be seen as a white band in the 0.8 M sucrose

step. Remove the upper part of the gradient carefully with a Pasteur pipet. Collect
the white band from the gradient, try to keep the volume small, normally between
250-400 µ L.

11. Dialyze the vesicles against Buffer B for 2-3 d with 2 buffer changes per day.

12. Analyze the vesicles by protein and lipid determination and by EM.

3.3. Vesicle Analysis
3.3.1. Protein Determination

This method allows the determination of protein even at a high lipid/
protein ratio.

1. Ideal protein quantity in the sample: 10-20 µ g.

2. Make a calibration curve consisting of 0, 20, 40, 60, 80, 100 µ L of the BSA standard.

3. Add water to 1000 mL.