Experiment Protocol 003S
Immobilization of ligands (compounds with phenol groups or NH2 groups) on epoxy
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 ligands at four various concentrations, i.e. 0 mM, 2 mM, 10 mM, and 50 mM when immobilizing ligands on epoxy
beads.

1. Materials
1.1 Beads and Ligands (Compounds)
・Epoxy beads (TAS8848N1110):4 mg (Functional groups : Approx. 200 nmol/mg)
・Ligands: Approx.8 mg
1.2 Reagents
・N,N-Dimethylformamide (DMF) 6 mL・Potassium carbonate (K2CO3) M.W. 138.21 20 mg
・Methanol 2.8 mL
1.3 Apparatus
・Micro centrifuge
・Incubator
・Rotator
・Ultrasonic dispersing device
We have performed operation checks with an ultrasonic homogenizer:VP-15S with a cup horn(TAITEC), and an ultrasonic dispersing device:TA4905 (Tamagawa Seiki).
2. Method
2.1 Outline
The following is a schematic view of ligand immobilization. Both phenolic OH groups and NH2
groups can be immobilized in the same method. Refer to the next section 2.2 “Procedures” fordetails.
LigandOH

Phenol groups

HNOH

OH
OO

O

NH2 groups

HNO

LigandO

OOH

OH
LigandO

OH

Phenol groups ligand immobilized beads

OHO

H
N

K2CO3

Epoxy beads
OHOH

O

K2CO3NH2HNOH
O

O

NH

LigandOH

Epoxy beads
NH2 groups ligand immobilized beads

2.2 Procedures
1) Dissolve ligands (compounds) in DMF, and prepare 400 µL of 50 mM ligand solution.
2) Add 1 mg of epoxy beads (TAS8848N1110) into each of four 1.5 mL micro-tubes.
3) Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
4) Add 200 µL of DMF, and disperse the beads with an ultrasonic device.
5) Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
6) Repeat the above 4) to 5) two more times. (Wash the beads three times in total.)7) Add DMF and the prepared ligand solution, and disperse the beads with an ultrasonic device.
(Refer to the table on the next page.)8) Add potassium carbonate of 10 mol times to ligands (e.g. 14 mg of potassium carbonate to 50
mM of ligands), and mix them with an ultrasonic device.
(Or put the measured potassium carbonate into another tube, and add ligands and beads
solution into the tube)

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Experiment Protocol 003S

9)
10)
11)
12)

13)
14)
15)
16)
17)
18)
19)

Concentration

(mM)

0

2

1050

Epoxy beads(mg)

1

1

1

1DMF(µL)

200

192

169

050 mM ligand(µL)

0840200

Potassium carbonate

(mg)

00.56

2.8

14Total(µL)

200

200

200

200

Note: If the ligand solution is added directly to the beads, the concentration could be
locally raised. Add, therefore, the ligand solution after adding the DMF to thebeads.
React them for 16 to 20 hours (overnight) at 60℃ by using a rotator in an incubator. (Thepotassium carbonate is not dissolved.)
Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
Add 200 µL of 50% DMF, and disperse the beads with an ultrasonic device.
Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
(As beads collect in a middle layer of the solution, discard the solution only in an upperlayer.)
Repeat the above 11) to 12) one more time. (Wash the beads twice in total.)
Add 200 µL of ultrapure water, and disperse the beads with an ultrasonic device.
Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
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 the supernatant.
Repeat the above 16) to 17) two more times. (Wash the beads three times in total.)
Resuspend in 40 µL of 50% MeOH, and store 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.)

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Experiment Protocol 003S・Recover beads dispersed in DMF or 50% MeOH by centrifugation because the magnetic separation
takes a longer time.
・Use DMF which is hydrated with a molecular sieve, or a low-moisture solvent.
・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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TAMAGAWA SEIKI CO., LTD.
PRD001479W00 20170710