Experiment Protocol 008
Immobilization of ligands (compounds with NH2 groups) on COOH beads
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 the ligand concentrations at four steps, i.e. 0mM, 0.1mM, 0.3mM, and 1mM when immobilizing ligands on COOH
beads.

1. Materials
1.1 Beads and ligands （compounds）
・COOH beads （TAS8848N1140）:10 mg （Functional groups：Approx. 200nmol/mg）
・Ligands: Approx. 2 mg
1.2 Reagents
・N,N’-dimethylformamide （DMF） 18 mL
・N-hydroxysuccinimide （NHS） M.W. 115.09 100 mg
・1-etyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride （EDC・HCl）
M.W. 191.70 100 mg
・Amino ethanol M.W. 61.08 200 µL
・Triethylamine M.W. 101.19
・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.
1) Active esterification

COOH beads

NHS beads

2) Ligand immobilization

NHS beads

NH2 grops ligand immobilized beads

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2.2 Procedures
2.2.1 Active esterification
1) Dissolve succinimide in DMF, and prepare 500 µL of 1M succinimide solution.
2) Add COOH beads (TAS8848N1140) into a 1.5 ml micro-tube. Repeat this one more time.
(total 10 mg)
3) Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
4) Add 500 µL of DMF, and disperse the beads.
5) Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
6) Repeat the above 4) - 5) two more times. (Wash the beads three times in total.)7) During the washing, add 38.4 mg (200 µmol) of EDC・HCl into another 1.5 mL micro-tube.
8) After DMF washing, add 800 µL of DMF, and disperse the beads.
9) Add 200 µL of the prepared 1 M succinimide solution, and mix them.
10) Transfer the total volume of the above 9) to the micro-tube of the above 7), and mix them.
（mg）
（µL）
（µL）
（mg）
（µL）

COOH beads
DMF
1M succinimide
EDC・HClTotal

5800
20038.4100011) React them for two hours at room temperature by using a micro tube mixer.
12) Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
13) Add 500 µL of DMF, and disperse the beads.
14) Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
15) Repeat the above 13) - 14) four more times. (Wash the beads five times in total.)16) Disperse the beads into 100 µL DMF each. (Concentration of NHS beads：2.5 mg/50 µl)
2.2.2 Immobilization of ligands
1) Dissolve ligands (compounds) in DMF, and prepare 1 mL of 1 mM ligand solution.
2) Dissolve amino ethanol in DMF, and prepare 3 mL of 1M amino ethanol solution.
3) Add 2.5 mg (50 µL) of the NHS beads to each of the four micro-tubes. Centrifuge at 15,000rpm for five minutes at room temperature, and discard the supernatant.
4) Add 200 µL of DMF to each of them, and disperse the beads. Centrifuge at 15,000 rpm forfive minutes at room temperature, and discard the supernatant.
5)
Add DMF and the prepared ligand solution, and disperse the beads with an ultrasonicdispersing device.
Concentration
(mM)
00.1
0.3
1
NHS beads
(mg)
2.5
2.5
2.5
2.5
DMF(µL)500
450
350
01mM ligand(µL)
050150500Total(µL)
500
500
500
500
Note 1: If the ligand solution is directly added to the beads, the concentration could belocally raised. Add, therefore, the ligand solution after adding the DMF to thebeads.
Note 2: When ligands are hydrochloride, add twice mole tri-ethyl amine to them. In thiscase, it will be easier to prepare 4 mM triethylamine and 2 mM ligand solution,and mix them in equal volume.

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Experiment Protocol 008
6)
7)
8)
9)
10)
11)
12)
13)
14)

React them for seventy minutes at room temperature by using a micro tube mixer.
Centrifuge at 15,000 rpm for five minutes at room temperature, and transfer the supernatant
to a fresh micro-tube. (Supernatant A)
Add 500 µL of amino ethanol to the remaining beads each, and disperse the beads with anultrasonic dispersing device.
React them for two hours at room temperature by using a micro tube mixer.
(Masking of ligand-non-binding carboxyl groups)
Centrifuge at 15,000 rpm for five minutes at room temperature, and transfer the supernatant
to a fresh micro-tube. (Supernatant B)
Add 500 µL of 50% MeOH, and disperse the beads with an ultrasonic dispersing device.
Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
Repeat the above 11) - 12) two more times. (Wash the beads three times in total.)
Disperse the beads in 100 µL of 50% MeOH, and store them at 4℃. (Concentration of ligand
immobilized beads:0.5 mg/20 µL）

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 DMF or 50% MeOH not by magnetic separation but by centrifugation
because the magnetic separation takes a longer time.
・Use DMF which is hydrated with a molecular sieve, or a low-moisture solvent. If the solvent
contains moisture, succinimide may be liberated from beads, and ligands are not properly
immobilized on the beads.
・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.
・You can investigate the following by determining quantity of succinimide in the supernatant A and
B by means of HPLC.
(Refer to Experiment Protocol 201 for the method.)
A：The amount of immobilization of ligands(The amount of succinimide liberated when immobilizing ligands.)

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Experiment Protocol 008
B：The amount of NHS groups on which ligands are not immobilized.
(The amount of succinimide liberated when masking.)
A+B：The amount of NHS groups of beads
・You can enhance the reactivity by adding tri-ethyl amine even when ligands are not hydrochloride if
the volume of the immobilization is small.
・Use DMSO when ligands have low solubility in DMF. However, when quantify the amount ofsuccinimide by HPLC using Experiment Protocol 201, decrease the concentration of DMSO down to
10% or less, for avoiding the peaks of DMSO overlap the peaks of succinimide.

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