Reconstitution of Functional Integrin into Phospholipid Vesicles and Planar Lipid Bilayers - Methods in Molecular Biology: Extracellular Matrix Protocols - page 73

7. Four-step sucrose gradient with 0.4 M, 0.8 M, 1.2 M and 2 M sucrose: prepare

stock solutions of 0.4 M, 0.8 M, 1.2 M , and 2 M sucrose in Buffer A. Take a clear
4-mL centrifugal tube and add 850 µ L 2 M sucrose, then overlay with 850 µ L

1.2 M and 850 µ L 0.8 M, followed by 600 µ L of 0.4 M sucrose.

2.3. Vesicle Analysis
2.3.1. Protein Determination
1. BSA standard, 0.2 mg/ml

2. 0.15% Na-deoxycholate: dissolve 15 mg Na-deoxycholate in 10 mL distilled

H 2 O, store at -20 ° C.

3. 72-100 % (w/v) TCA: Dissolve 100 g TCA (72-100%) in distilled water to a

final volume of 100 mL.

4. 1% (w/v) CuSO 4 , store at 4 ° C.

5. 2% (w/v) Na-K-tartrate, store at 4 ° C.

6. 10% (w/v) SDS, store at room temperature.

7. 0.8 M NaOH, store at room temperature.

8. Solution A: Dissolve 200 µ g Na 2 CO 3 in 1600 µ L H 2 O, add 200 µ L 1% CuSO 4 and

200 µ L 2% Na-K-tartrate, add 2 mL 10% SDS, 2 mL 0.8M NaOH, and 10 mL H 2 O.

9. Solution B: Dilute Folin-Ciocalteau-reagent (Merck, Darmstadt, Germany) 1:6

with distilled water.

2.3.2. Phosphorus Determination
1. Perchloric acid, not exceeding 72%.

2. 10% ascorbic acid, freshly prepared and stored at -20 ° C at the latest for 1 mo.

3. Molybdate reagent: To 2.2 g (NH 4 ) 6 Mo 7 O 24 *4 H 2 O add 14.3 mL H 2 SO 4 ,

95-98% and then slowly add distilled water to 1000 mL. Prevent from
exposure to light.

4. X100 phosphate standard, 40 m M ,: To 712.0 mg Na 2 HPO 4 *2H 2 O add distilled

water to a final volume of 100 mL. Dilute to X1 phosphate standard, 400 µ M ,

standard, store at -20 ° C.

5. Phosphate-free, disposable glass tubes (borate glass).

2.3.3. Electron Microscopy
1. 2% (v/v) Na-phosphotungstate in distilled water, adjusted to pH 7.0, sterile fil-

tered through a 0.22- µ m filter prior to use.

2. Collodion carbon grids, glow discharged for 30-45 s in a glow discharger.

2.4. Vesicle Fusion
1.

Buffer C: 20 m M Tris-HCl pH 7.4, 150 m M NaCl, 1 m M CaCl 2 , 1 m M MgCl 2 ,
1 m M MnCl 2 . Dissolve MnCl 2 directly before the buffer is used ( see Note 5 ).

2. Detergent for cleaning the quartz slides: Linbro 7X-PF (ICN Flow Biochemicals,

Costa Mesa, CA), diluted 1:6 with distilled water, can be used five times for washing.

3. Quartz slides.