Experiment Protocol 106
Immobilization of antibodies or proteins on Epoxy beads
1. Materials
1.1 Beads and Ligands (Antibodies)・Epoxy beads (TAS8848N1110): 1 mg (Functional groups：Approx. 200 nmol/mg)
・Antibodies or Proteins: approximately 50 µg
1.2 Reagents・2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)
・Sodium hydroxide ・Potassium chloride ・Ethylenediaminetetraacetic acid (EDTA)
・Glycerol
Composition of protein immobilization buffer
10mM HEPES-NaOH (pH7.9)
Composition of protein immobilized beads wash/storage buffer
10mM HEPES-NaOH (pH7.9)
50mM KCl
1mM EDTA
10% glycerol
1.3 Apparatus
・Micro high-speed cold 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.

Epoxy beads

Antibody (protein) immobilized beads

2.2 Procedures1)

Prepare protein immobilization buffer and washing/storing buffer2)

Dilute antibodies with protein immobilization buffer, and prepare 50 µg/50 µL antibody
solution by 50 µL or more. (Prepare the solution slightly more than 50 µL so that you can fully
secure 50 µL.)
Add 1 mg of epoxy beads (TAS8848N1110) into a 1.5 mL micro-tube.
Centrifuge at 15,000 rpm for five minutes at 4℃, and discard the supernatant.
Add 50 µL of protein immobilization buffer, and disperse the epoxy beads with an ultrasonicdevice.
Centrifuge at 15,000 rpm for five minutes at 4℃, and discard the supernatant.

3)
4)
5)
6)

1

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Experiment Protocol 106
7)
8)
9)
10)
11)
12)
13)
14)
15)

Repeat the above 5) to 6) two more times. (Wash the beads three times in total.)
Add 50 µL of protein immobilization buffer, and disperse the beads with an ultrasonic device.
Add 50 µL of antibody solution or protein solution.
React for 16 to 20 hours (overnight) at 37℃ by using a micro tube mixer.
Centrifuge at 15,000 rpm for five minutes at 4℃, and transfer the supernatant to a flesh
tube. (For protein quantification)
Add 50 µL of protein immobilized beads wash/storage buffer, and disperse the beads by themanual agitation.
Centrifuge at 15,000 rpm for five minutes at 4℃, and discard the supernatant.
Repeat the above 12) to 13) two more times. (Wash the beads three times in total.)
Resuspend in 200 µL of protein immobilization beads washing/storing buffer, and store at 4℃.
(Concentration of antibody immobilized beads：0.1 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.)・When dispersing the beads after immobilizing antibodies, disperse them by the manual agitation.
When you cannot disperse the beads easily, disperse them in a short time by using an ice-cold
ultrasonic homogenizer or ultrasonic washer.
・Recover the beads not by magnetic separation but by centrifugation.
・The amount of proteins and antibodies immobilized on beads can be calculated from protein
quantification (Bladford method or SDS-PAGE) of the transferred supernatant. It can also becalculated directly from protein immobilized beads by BCA method.
・ When you want to increase the volume of proteins immobilized on beads, increase the volume of theproteins to be added.

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PRD001501W00 20170710