Experiment Protocol

109

Immobilization of ligands (alkyne structure compounds) on azide beads using click
chemistry reaction
For screening, you need, first of all, to optimize the amount of immobilization of ligands on beads. You can change theamount of immobilization of ligands by changing the concentration of ligands. This experiment protocol shows a method toimmobilize ligands at four various concentrations, i.e. 0µM, 5µM, 25µM, and 125µM when immobilizing ligands on azide beads.

1. Materials
1.1 Beads and Ligands (Compounds)・Azide beads （TAS8848N1160）:10 mg （Functional groups：Approx. 100 nmol/mg）
・Ligands: Approx. 0.1 mg
1.2 Reagents・t-butyl alcohol (t-BuOH) 12 mL・Dimethylsulfoxide (DMSO) 3 mL・Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine（TBTA） M.W. 530.63 2.7 mg
・Copper(Ⅱ)sulfate（CuSO4） M.W. 159.61 16 mg
・(＋)-Sodium L-ascorbate M.W. 198.11 20 mg
・Methanol （MeOH） 4 mL
1.3 Apparatus
・Micro centrifuge
・Micro tube mixer （TOMY MT-360, etc.）
・Ultrasonic dispersing device
We have performed operation checks with an ultrasonic homogenizer:VP-15S with a cup horn（TAITEC）, or an ultrasonic dispersing device:TA4905 （Tamagawa Seiki）.
2. Method
2.1 Outline
The following is a schematic view of ligand immobilization. Refer to the next section 2.2“Procedures” for details.

Azide beads

Ligand immobilized beads

2.2 Preparation of solutions
1)
Prepare 15 mL of t-BuOH/DMSO solution by mixing 12 mL of t- BuOH with 3 mL ofDMSO(t-BuOH : DMSO = 4:1)
※Since t-BuOH has a low freezing point (25.7 ℃), if the t-BuOH is solidified, please bedissolved before use.
2) Dissolve ligands (compounds) in t-BuOH/DMSO solution, and prepare 200 µL of 500 µM
ligand solution.
3)
Dissolve 2.7 mg of TBTA in 1mL of t-BuOH/DMSO solution, and prepare 1mL of 5 mM
TBTA solution. Add 190 µL of t-BuOH/DMSO solution to 10 µL of 5 mM TBTA, and prepare
200 µL of 250 µM TBTA solution.
4)
Dissolve 16 mg of CuSO4 in 1 mL of ultrapure water, and prepare 1 mL of 100 mM CuSO4solution. Add 190 µL of ultrapure water to the 10 µL of 100 mM CuSO4 solution, and prepare
200 µL of 5 mM CuSO4 solution.

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Experiment Protocol5)

6)

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Dissolve 20 mg of (＋)-Sodium L-ascorbate in 1 mL of ultrapure water, and prepare 1 mL of
100 mM (＋)-Sodium L-ascorbate solution. Add 190 µL of ultrapure water to 10 µL of the
100 mM (＋)-Sodium L-ascorbate solution, and prepare 200 µL of 5 mM (＋)-SodiumL-ascorbate solution.
Prepare 8 mL of t-BuOH/DMSO/ultrapure water solution by mixing 4 mL of thet-BuOH/DMSO solution prepared in the above 1) with 4 mL of ultrapure water.
(t-BuOH/DMSO solution: ultrapure water = 1:1)

2.3 Ligand immobilization1)
Add 2.5 mg of azide beads into each of four micro-tubes. Centrifuge at 15,000 rpm for fiveminutes at room temperature, and discard the supernatant.
2)
Add each reaction solution in the order as shown below, from the top solution Azide beads tothe bottom Sodium ascorbate. In this case, after adding 250 µM of TBTA, disperse the beads
with an ultrasonic device. Then, add the remaining solutions.

3)4)Concentration（µM）

0525125Azide beads

（mg）

2.5

2.5

2.5

2.5

t-BuOH/DMSO

（µl）

250

240

200

0500µM ligands（µl）

0525125

250µM TBTA（µl）

0525125

Ultrapure water

（µl）

250

240

200

05mM CuSO4（µl）

05251255mM (＋)-Sodium ascorbate（µl）

0525125Total

（µl）

500

50050050010)

React for 16 to 20 hours at room temperature by using a micro tube mixer.
Centrifuge at 15,000 rpm for five minutes at room temperature, and discard thesupernatant.
Add 500 µl of t-BuOH/DMSO/ultrapure water solution, and disperse the beads with anultrasonic device.
Centrifuge at 15,000 rpm for five minutes at room temperature, and discard thesupernatant.
Repeat the above 7) to 8) two more times. (Wash the beads with t-BuOH/DMSO/ultrapure
water solution three times in total.)
Add 500 µL of 50% MeOH, and disperse the beads with an ultrasonic device.
Centrifuge at 15,000 rpm for five minutes at room temperature, and discard thesupernatant.
Repeat the above 10) to 11) two more times. (Wash the beads three times in total.)

11)

Disperse the beads in 100 µL of 50% MeOH, and store at 4℃. (Concentration of ligand5)
6)
7)
8)
9)

immobilized beads: 0.5 mg/20 µL)

2

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3. Supplements・Beads are easily dispersed by using an ultrasonic dispersing device. But if you do not have such adevice, they are dispersed by using an ultrasonic washer, or by the manual agitation. In the manual
dispersion method, the bottom of a micro-tube is glided over an uneven surface (side of plastic test
tube rack in this case) creating turbulence through the collisions. (see left side picture below)
Please make sure to use well-constructed tubes with the caps tightly secured in order to prevent
leakage/breakage. Use of cap lock is recommended in order to prevent leakage. (see right sidepicture below).
For more information, please visit FG beads web site and see the movie of the method.
(Please click : http://www.magneticnanoparticle.jp/en/htdocs/technique/affinity.html for movingpictures.)・Recover beads dispersed in t-BuOH, DMSO, or 50% MeOH not by magnetic separation but by
centrifugation because the magnetic separation takes a longer time.
・Although we recommend using 50% MeOH for storing ligand immobilized beads in view of thedecrease of dispersibility of beads due to immobilization of hydrophobic compounds, you cansatisfactorily use ultrapure water, too.

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