Experiment Protocol 005S
Immobilization of ligands (carboxylic compounds) on NH2 beads (1) A method ofusing HOSu
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 protocol shows a method to immobilize
ligands at four various concentrations, i.e. 0 mM, 0.4 mM, 2 mM, and 10 mM when immobilizing ligands on NH2 beads.

1. Materials
1.1 Beads and Ligands (compounds)
・NH2 beads (TAS8848N1130):4 mg (Functional groups：Approx 200nmol/mg)
・Ligands: Approx. 2 mg
1.2 Reagents
・N,N’- Dimethylformamide (DMF) 10 ml
・N- Hydroxysuccinimide (HOSu) M.W. 115.09 2 mg
・1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) M.W. 155.24
2 mg (Peptide Institute 1020, etc.)
・Acetic anhydride M.W. 102.09 160 µl
・Methanol (MeOH) 2 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), 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.

Activated ligands

NH2 beads

COOH ligand immobilized beads

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Experiment Protocol 005S
2.2 Procedures
1) Dissolve ligands (compound) in DMF, and prepare 4 µmol/200 µL (20mM) solution.
(Final 10 mM chemical compound)2) Dissolve succinimide in DMF, and prepare 40 µL of 200 mM succinimide solution.
3) Dissolve EDC in DMF, and prepare 40 µL of 200 mM EDC solution.
4)

Add 160 µL of DMF, 20 µL of 200 mM succinimide solution, and 20 µL of 200 mM EDC
solution to 200 µL of 20 mM ligand solution as below, and mix for two hours at room
temperature by using a micro tube mixer. (Mix them in equal mol.)

5)
6)
7)
8)
9)
10)
11)
12)

20mM ligand (compound)(µL)

200 (10 µmol)

200mM succinimide(µL)

20(10 µmol)

200mM EDC(µL)

20(10 µmol)DMF(µL)

160

Total(µL)

400

Add 1 mg of NH2 beads (TAS8848N1130) into each of four 1.5 mL micro-tubes.
Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
Add 500 µL of DMF, 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 7) to 8) two more times. (Wash the beads three times in total.)
Add DMF for each ligand immobilization concentration.
Add the prepared activated 10 mM ligand solution, and disperse the beads with an ultrasonicdevice as below.
React for 16 to 20 hours at room temperature by using a microtube mixer.
Concentration

(mM)

00.4

2

10

NH2 beads(mg)

1

1

1

1

DMF(µL)

200

192

160

0

Activated 10mM ligand

(µL)

0

8

40

200

Total(µL)

200

200

200

200

Note: If the ligand solution is added directly to the beads, the concentration could be locallyraised. Add, therefore, the ligand solution after adding the DMF to the beads.
13)
14)
15)
16)
17)
18)
19)

Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
Add 200 µL of DMF, 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 14) to 15) two more times. (Wash the beads three times in total.)
Resuspend in 160 µL of DMF by ultrasonic waves.
Add 40µL of acetic anhydride. (20% acetic anhydride)
Mix for two hours at room temperature by using Microtube Mixer.
(Masking of ligand-unreacted amino groups)20) Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
21) Add 200 µL of DMF, and disperse the beads with an ultrasonic device.

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Experiment Protocol 005S
22) Centrifuge at 15,000 rpm for five minutes at room temperature, and discard the supernatant.
23) Repeat the above 21) to 22) two more times. (Wash the beads three times in total.)
If the chemical compound has functional groups to be acetylated by acetic anhydride such as anOH group, perform a deacetylation process, after masking, by resuspending in 200 µL of 0.1 M
sodium hydroxide, and by mixing for 30 minutes at room temperature by using Microtube Mixer.
After then, repeat the resuspension by centrifugation and ultrasonic waves, and wash with 200µL of ultrapure water three times.
24)
25)
26)
27)

Add 200 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 24) to 25) 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.)・ 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.
・ To Mask of ligand-unreacted amino groups use 20% acetic anhydride for two hours at room
temperature, or 1% acetic anhydride for 16 to 20 hours (overnight) at room temperature.

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