Experiment Protocol 102
Immobilization of His-Tag proteins on Ts beads
For screening, you need, first of all, to optimize the amount of immobilization of proteins on beads. You can change theamount of immobilization of proteins by changing the concentration of proteins. This experiment protocol shows a method toimmobilize proteins at four various concentrations, i.e. 0µM（0nmol/mg）, 2µM（0.4nmol/mg）, 10µM（2nmol/mg）, and 50µM
（10nmol/mg） when immobilizing proteins on Ts beads

1. Materials
1.1 Beads and Ligands (His-Tag proteins)・Ts beads (TAS8848N1150): 10mg（2.5mg/condition）
・His-tag proteins: Approx. 2 mg
1.2 Reagents・2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid(HEPES)
・Sodium hydroxide ・Potassium chloride ・Ethylenediamine tetraacetic acid (EDTA) ・Glycerol
・Tris(hydroxymethyl)aminomethane M.W. 121.14 ・Hydrochloric acid
Composition of protein immobilization buffer
10mM HEPES-NaOH(pH7.9)
50mM KCl
1mM EDTA
10% glycerol
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 cuphorn(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 for details.

OHH
NOHOOOOH

Ts beads

HNO
SO+NProtein

His-Tag proteins

His-Tag protein immobilized beads

2.2 Procedures
1)
2)

3)
4)

Prepare protein immobilization buffer.
Dilute proteins with protein immobilization buffer, and prepare 500 µl of protein solution foreach concentration, 0 µM, 2 µM, 10 µM, and 50 µM.
※We recommend dialyzing the protein immobilization buffer when the buffer composition ofprotein solution differs.
Dissolve tris(hydroxymethyl)aminomethane in ultrapure water, and prepare 3mL of 1M tris
(hydroxymethyl)aminomethane (pH8.0). (Adjust the pH with hydrochloric acid.)
Add 2.5 mg of Ts beads (TAS8848N1150) into each of four 1.5 ml micro-tubes.

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Experiment Protocol 1025)

Centrifuge at 15,000 rpm for five minutes at 4℃, and discard the supernatant.

6)

Add 500 µL of protein immobilization buffer, and disperse the Ts beads with an ultrasonic

7)
8)

device.
Centrifuge at 15,000 rpm for five minutes at 4℃, and discard the supernatant.
Repeat the above 6) to 7) two more times. (Wash the beads three times in total.)9)

Add 500 µL of the prepared protein solution of each concentration, and disperse the beads
with an ultrasonic dispersing device.

10)

React for 16 to 20 hours (over night) at 4℃ by using a micro tube mixer.

11)

Centrifuge at 15,000 rpm for five minutes at 4℃, and transfer the supernatant to a fresh
micro-tube. (For protein quantification)

12)

14)

Add 500 µL of 1M tris hydroxymethyl amino methane (pH8.0) to the remaining beads, anddisperse the beads.
React for 16 to 20 hours (over night) at 4℃ by agitating with a micro tube mixer. (Masking ofprotein-non-binding tosyl groups)
Centrifuge at 15,000 rpm for five minutes at 4℃, and discard the supernatant.

15)

Add 500 µL of protein immobilization buffer, and disperse the Ts beads with an ultrasonic16)
17)

device.
Centrifuge at 15,000 rpm for five minutes at 4℃, and discard the supernatant.
Repeat the above 15) to 16) two more times. (Wash the beads three times in total.)

18)

Disperse the beads in 500 µL of protein immobilization buffer, and store them at 4℃.

13)(Concentration of His-Tag protein immobilized beads:0.5 mg/100 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.htmlfor movingpictures.)

・The amount of immobilized His-Tag proteins can be calculated from protein quantification (Bladford
method or SDS-PAGE) of the transferred supernatant. It can also be calculated directly fromprotein immobilized beads by BCA method.

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Experiment Protocol 102
・When you want to increase the amount of His-Tag proteins immobilized on beads, increase theamount of the proteins to be added.
・When beads and proteins attach to the wall of a micro-tube, disperse them as much as possible.

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