Experiment Protocol 014
Immobilization of ligands (compounds with NH2 groups) on NHS 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, 0.1 mM, 0.3 mM, and 1 mM when immobilizing ligands onNHS beads.

1. Materials
1.1 Beads and Ligands (Compounds)
・NHS beads (TAS8848N1141):10 mg (Functional groups : Approx. 200 nmol/mg)
・Ligands: Approx.2 mg
1.2 Reagents
・N,N-Dimethylformamide (DMF) 7 mL・Amino ethanol M.W. 61.08 200 µL ・Triethylamine M.W. 101.19 1 µL・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 acup horn(TAITEC), and 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.

NHS beads

NH2 group ligand immobilized beads

2.2 Procedures
1) Dissolve ligands (compound) in DMF, and prepare 1 mL of ligand solution.
2) Dissolve amino ethanol in DMF, and prepare 3 mL of amino ethanol solution.
3) Add 2.5 mg of NHS beads into each of four micro-tubes. Centrifuge at 15,000 rpm for fiveminutes at room temperature, and discard the supernatant.
4) Add 200 µL of DMF, and disperse the beads. Centrifuge at 15,000 rpm for five minutes at room
temperature, and discard the supernatant.
5) Add DMF and the prepared ligand solution, and disperse the beads with an ultrasonic device.

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Experiment Protocol 014

Concentration
NHS beads
DMF
1mM ligands
Total（mM）
（mg）
（µL）
（µL）
（µL）

02.5
500
0500

0.1
2.5
450
50
500

0.3
2.5
350
150
500

1
2.5
0500
500

Note 1: If the ligand solution is added directly 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 tri-ethyl amine and 2mM ligand solution,and mix them in equal volume.
6)

React them for seventy minutes at room temperature by using a micro tube mixer.

7)

Centrifuge at 15,000 rpm for five minutes at room temperature, and transfer the supernatant
to a fresh micro-tube. (Supernatant A)

8)

Add 500 µL of 1 M amino ethanol to the remaining beads each, and disperse the beads withanultrasonic dispersing device.

9)

React them for two hours at room temperature by using a micro tube mixer.
(Masking of ligand-non-binding carboxyl groups)

10)

Centrifuge at 15,000 rpm for five minutes at room temperature, and transfer the supernatant11)
12)
13)

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) to 12) two more times. (Wash the beads three times in total.)

14)

Resuspend 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 moving
pictures.)

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Experiment Protocol 014
・ 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.
・You can investigate the following by determining quantity of succinimide in the supernatant A andB by means of HPLC.
(Refer to Experiment Protocol 201 for the method.)
A : The amount of immobilization of ligands (The amount of succinimide liberated whenimmobilizing ligands.)
B : The amount of NHS groups on which ligands are not immobilized. (The amount ofsuccinimide liberated when masking.)
A+B : The amount of NHS groups of beads
・Use DMSO when ligands have low solubility in DMF. However, when quantify the amount of
succinimide by HPLC using Experiment Protocol 201, decrease the concentration of DMSO down to
10% or less, for avoiding the peak of DMSO overlap the peaks of succinimide.

3

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PRD001493W00 20170710