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<table class="sample_04">
<table class="sample_04">
<caption>Table 1. Breakdown of Samples</caption>
<tbody>
<tbody>
<caption>Table 1. Breakdown of Samples</caption>
<tr>
<tr>
<th>sample No.</th>
<th>sample No.</th>

Revision as of 19:43, 27 August 2014

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<body> PROTOCOL

<a href="#" onclick="HideCBox('CBoxBody1'); return false;" title="折りたたみ／復元">[show/hide]</a>

  Contents：

  • <a href=#1>1. Polymerization </a>
    • <a href="#1-1">1-1. Making the Monomer </a>
    • <a href="#1-2">1-2. Controlling polymerization </a>
    • <a href="#1-3">1-3. Putting the Monomer into a liposome </a>
    • <a href="#1-4">1-4. Polymerization </a>
      • <a href="#1-4-1">1-4-1. Polymerization in solution </a>
      • <a href="#1-4-2">1-4-2. Polymerization in a liposome </a>
    • <a href="#1-5">1-5. Deformation of a liposome </a>
  • <a href="#2">2. Receptor </a>
    • <a href="#2-1">2-1. Making the Receptor </a>
    • <a href="#2-2">2-2. Penetration into a liposome </a>
    • <a href="#2-3">2-3. Dimerization
      • <a href="#2-3-1">2-3-1. Dimerization in solution </a>
      • <a href="#2-3-2">2-3-2. Dimerization on a liposome </a>
    • <a name="1"><a href="#2-4">2-4. Emission of polymerization initiator by dimerization </a></a>
      • <a href="#2-4-1">2-4-1. Emission by dimerization in solution </a>
      • <a name="1-1"><a href="#2-4-2">2-4-2. Emission by dimerization on a liposome </a></a>

<a name="contents"> 1. Polymerization</a>


1-1. Making the Monomer

<tbody> </tbody>
Reagents (f. 60 µL)
Staple mix 17.5 µL
M 13 mp 18 ss 18.3 µL
TE¹ 18.2 µL
10 × tile buffer² 6.0 µL
<tbody> </tbody>
¹TE (f. 50 mL)
1 M Tris - HCl (pH 8.0) (f. 10 mM) 0.5 mL
0.5 M EDTA (f. 1 mM) 0.1 mL
MQ 49.4 mL
<tbody> </tbody>
²10 × tile buffer(f. 500 mL)
Mg (OAc) 2 (f. 100 mM) 10.73 g
1.0 M Tris-HCl (pH 7.5) (f. 200 mM) 100 mL
0.5 M EDTA (f. 10 mM) 10 mL
MQ up to 500 mL

Procedure

1) The reagents were mixed.

2) The mixture was annealed at 55 °C for 3 hours.

3) The mixture was analyzed by 1 % agarose gel electrophoresis.

4) The gel was stained by EtBr for 30 minutes.

5) The gel was taken photo by LAS - 4000.


<a name="1-2"> </a>



1-2. Controlling polymerization

<a name="1-3"> </a>



1-3. Putting the Monomer into a liposome

<tbody> </tbody>
Reagents (f.1420 µL)
Lipid mix (f. 0.5 mM)¹ 400 µL
Glucose (f. 200 mM) 500 µL
Emulsion mix² 520 µL
<tbody> </tbody>
¹Lipid mix (f. 3 mM)
POPC 12 mg
Paraffin 5 mL
<tbody> </tbody>
²Emulsion mix
Lipid mix (f. 0.5mM) 500 µL
Inner solution³ 10 µL
The Monomer 10 µL
<tbody> </tbody>
³Inner solution
HEPES (f. 50 mM) 250 µL
Sucrose(f. 400 mM) 250 µL
Pyranine 2 µL

Procedure

1) Take glucose (500 µL) into a tube as outer solution.

2) Lipid mix (0.5 mM) was added on the glucose solution carefully.

3) Emulsion mix was added on the top and rapidly centrifuged at 4 °C, 1800 rpm for 10 minutes.

4) Centrifuged again at 4 °C, 5000 rpm for 10 minutes.

5) The upper layer was removed and GUVs were collected using pipetman.

6) The liposome was stained by Nile red and observed with a confocal microscope.


<a name="1-4"> </a>

<a name="1-4-1"> </a>


1-4. Polymerization

  1-4-1. Polymerization in solution

<tbody> </tbody>
Reagents
Purified Monomer A 10 µL
Purified Monomer B 10 µL

Procedure

1) Monomers were made as shown in “Making DNA origami monomers”.

2) Each Monomer solution was purified by spin column.

3) The reagents was mixed.

4) The mixture was incubated at room temperature for 24 hours.

5) The mixture was analyzed by 1 % agarose gel electrophoresis (100 V, 80 min).

6) The gel was stained by EtBr for 30 minutes.

7) The gel was taken photo by LAS - 4000.

<a name="1-4-2"> </a>



  1-4-2. Polymerization in a liposome <a name="2"> </a>


<a name="2-1"> </a>

<a name="contents"> 2. Receptor</a>

2-1. Making the Receptor

<tbody> </body>
Reagents (f. 40 µL)
M 13 mp 18 ss 18 µL
Staple mix 18 µL
10 × OCK buffer¹ 4 µL
<tbody> </tbody>
¹10 × OCK buffer (f: 10 mL)
1 M Tris (HCl pH 7.5) (f. 50 mM) 500 µL
0.5 M EDTA-Na (pH 8) (f. 10 mM) 200 µL
1 M MgCl2 (f. 100 mM) 1 mL
5 M NaCl (f. 500 mM) 100 µL
MQ 8.2 mL

Procedure

1)the solutions were mixed.

2) The mixture was annealed at 47.5 °C for 4 hours.

3) The mixture was purified by spin column.

4) The mixture was analyzed by 1% agarose gel electrophoresis (100V, 40 min).


<a name="2-2"> </a>



2-2. Penetration into a liposome

Ⅰ. Preparation of GUVｓ

GUVs were made as shown in "Putting the Monomer into a liposome".

Ⅱ. Preparation of LUVｓ



Ⅲ. Hybridization of cholesterol oligomer with the Receptor

<tbody> </tbody>
Reagents (f. 7.5 µL)
Purified Receptor (0.05 µM) 6 µL
Cholesterol oligomer (8 µM) 1.5 µL

Procedure

The reagents were mixed and incubated at room temperature for 60 minutes.


Ⅳ. Flotation assay

<tbody> </tbody>
Reagents
The Receptor 100 µL
Cholesterol hybridized Receptor 100 µL
Liposomes (? mg/mL LUVs) 100 µL
2.25 M Sucrose buffer¹ 500 µL
1.6 M Sucrose buffer² 900 µL
150 mM KCl solution 100 µL
1 × Flotation buffer³ 600 µL
<tbody> </tbody>
¹2.25 M Sucrose buffer (f. ????)
HEPES - KOH (pH 7.6) (f. 50 mM) ?????
KCl (f. 100 mM) ?????
MgCl2 (f. 20 mM) ?????
Sucrose (f. 2.25 M) ?????
<tbody> </tbody>
²1.6 M Sucrose buffer (f. ????)
HEPES - KOH (pH 7.6) (f. 50 mM) ?????
KCl (f. 100 mM) ?????
MgCl2 (f. 20 mM) ?????
Sucrose (f. 1.6 M) ?????
<tbody> </tbody>
³1 × Flotation buffer (f. ?????)
HEPES - KOH (pH 7.6) (f. 50 mM) ?????
KCl (f. 100 mM) ?????
MgCl2 (f. 20 mM) ?????


Procedure

1) Each sample was mixed as shown below.

<tbody> </tbody>
Table 1. Breakdown of Samples
sample No. 1 2 3 4
Cholesterol hybridized Receptor 50 µL 50 µL — —
Receptor — — 50 µL 50 µL
Liposome 50 µL — 50 µL —
150 mM aqueous KCl solution — 50 µL — 50 µL
2.25 M Sucrose buffer 125 µL 125 µL 125 µL 125 µL

2) 225 µL of 1.6 M sucrose buffer was overlaid with 225 µL of sample mixture in centrifuge tubes (Beckman, cat#343778, 11 × 34 mm).

3) Centrifuge for 16 minutes at 100 krpm at 4 ℃ using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).

4) 150 µL of supernatant was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 150 µL of 1 × Flotation buffer (Fraction 4).

5) Fraction 1-4 of each sample were analyzed by 1 % agaraose gel electrophoresis (100V, 1 hour).

6) The Intensity of fluorescence of NileRed (Liposomes) was measured with fluorescence spectrophotometer (JASCO, FP-6500) to investigate the existence of liposomes in each Fraction.

7) The radiuses of liposome of each fraction were measured with DLS (Viscotek, 802 DLS).


<a name="2-3"> </a>

<a name="2-3-1"> </a>

2-3. Dimerization

  2-3-1. Dimerization in solution

<tbody> </tbody>
Reagents
Receptor E
Receptor A
Thrombin (1.09µM)
Loading-dye

Procedure

1) Mix the solutions, and incubated 1 hour at room temperature.

<a name="2-3-2"> </a>

2) Analyzed by 1% agarose gel electrophoresis (100V, 80 min).


  2-3-2. Dimerization on a liposome

<a name="2-4"> </a>

<a name="2-4-1"> </a>

2-4. Emission of polymerization initiator by dimerization

  2-4-1. Emission by dimerization in solution

<a name="2-4-2"> </a>

  2-4-2. Emission by dimerization on a liposome



















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