Experiment Protocol 201
Quantifying the amount of ligand immobilization by HPLC (High Performance
Liquid Chromatography)
Chromatography)
When screening target proteins of ligands, you may need to quantify the amount of ligand immobilization on beads. This
experiment protocol describes the method to quantify the amount of immobilization of ligands with NH2 groups on COOH
beads and NHS beads.

1. Materials
1.1 Supernatant A and B to immobilize on NHS beads (Refer to Experiment Protocol 008 and 014)
014)・Supernatant A(NHS contained solution centrifuged when immobilizing ligands)
・Supernatant B(NHS contained solution centrifuged when masking )1.2 Reagents
Reagents
・N-hydroxysuccinimide (NHS) M.W. 115.09
・N,N’-dimethylformamide (DMF)
・Ammonium acetate M.W. 77.08
・Acetic acid M.W. 60.05
・Acetonitrile (For High Performance Liquid Chromatography)
1.3 Apparatus
・Filter unit : Steritop (MILLIPORE SCGPT05RE)
・Micro-tube filter : Ultrafree-MC (MILLIPORE UFC30LG00)
・High Performance Liquid Chromatography (HPLC)(Waters 2695, 2998)
・Symmetry column 5µm C18 4.6×250mm stainless (Waters WAT054275)・Micro centrifuge
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Experiment Protocol 201
2. Method
2.1 Preparation
Preparation of reagent solutions1)
Preparation of 0.2 M ammonium acetate solution
1. Add 0.605 g of acetic acid into a 50 ml tube, and dilute this with ultrapure water in ameasuring cylinder to 50 ml total. (0.2 M acetic acid)
2. Add 7.708 g of ammonium acetate into a 500 ml beaker, and dilute this with ultrapure waterin a measuring cylinder to 400 ml total.
3. Add 0.2 M acetic acid until the pH of the solution becomes 5.70. (Approx. 40 ml)
4. Transfer the solution into a 500 ml measuring cylinder, and dilute this with ultrapure water
to 500 ml total.
5. Filter the solution (by using a filter unit), and store it at 4℃.
2)Preparation of 10 mM ammonium acetate solution：Mobile phase
Dilute 50ml of 0.2 M ammonium acetate solution in a measuring cylinder to 1l total.
3)

Preparation of standard curve for succinimide (NHS)
100 mM : 1M item used for immobilizing HOSu: 100 µl + DMF 900 µl
10 mM : 100mM 100 µl + DMF 900 µl
1 mM : 10mM 100 µl + DMF 900 µl
100 µM : 1mM 100 µl + DMF 900 µl
10 µM : 100µM 100 µl + DMF 900 µl
0 mM : DMF only
● Use 10 mM or lower only for standard curve.

4)

Preparation of buffer to dilute a sample
Prepare the solution with the buffer composition when starting the gradient.
Buffer :10 mM ammonium acetate (47 ml) (94% of the total)+ acetonitrile (3 ml) (6% of the total)
2.2 Preparation
Preparation of a sample1)
Mix 20 µl of a sample (standard curve, supernatant A and B) with180 µl of buffer to dilute thesample.
2)
Perform filtering to prevent beads being mixed into the HPLC. (For each 200µl：Use a micro
tube filter, and centrifuge at 5000×g for 1 min)3)
Add 150 µl of the filtrate into each vial, cover the vial, and install it to an auto-sampler of theHPLC.
2.3 Preparation of HPLC and programming1)
Switch on the HPLC (HPLC main body, column oven, and PC), and clean the passages.
2)
Install the column. (by pouring the solvent. Be careful about the direction of the arrow.
Confirm that liquid flows out.)
3)
Perform the buffer displacement in the column under the condition of 94% of 10mM
ammonium acetate and 6% of acetonitrile. (Wait until pressure stabilizes: approx. 1350psi)
4)
Perform the programming.
↓ Start up HPLC control software (Empower)↓ Set up the wizard (Capacity of a sample to be analyzed: 50 µl, the number of samples, etc.)↓ Set up the method (Gradient conditions: Fig. 1, Column: 40℃)

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Experiment Protocol 201
Method：Gradient 40minutes (Including washing processes)
Time(minute)

Flowvelocity(ml/min)

1012202240

11111110 mMammoniumacetate (%)
9460
20209494

Acetonitrile (%)

Curve

64080
80
6666666Gradient 40 min
1009080

Rate (%)

70
60
50

10mM Ammonium Acetate
Acetonitrile

40
30
20
10005101520
25Time (min)

30

3540

Fig. 1 Change of ammonium acetate solution mixing ratio of gradient 40 minutes
2.4
2.4 Analysis
1)
Select the wizard and the method you set up, and start the measurement.
2)
After the measurement, analyze at 260nm, and convert the peak area into a quantitativevalue.
3)
Wash the column with 100% acetonitrile (1ml/min) for one hour.
2.5
2.5 Data analysis
1)
From the peak area of a sample of supernatant A, calculate the amount of succinimide
separated when immobilizing the ligands.
2) Obtain the actual amount (µM) of ligand immobilization by subtracting, from the above 1),
the amount of succinimide when the amount of ligands to be added is 0 mM (ligands are notimmobilized) as the background. (Convert into mol/mg.)
3)
From the peak area of a sample of supernatant B, calculate the amount of succinimide
separated when masking.

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Experiment Protocol 2014)

5)

Obtain the amount (µM) of carboxyl groups on which ligands are not immobilized bysubtracting, from the above 3), the amount of succinimide of a sample of supernatant B
without masking as the background. (Convert into mol/mg.)
Obtain the amount of surface carboxyl groups of carboxylated beads by totaling the amount ofligand immobilization (A) obtained in the above 2) and the amount of carboxyl groups onwhich ligands are not immobilized (B) obtained in the above 4). (Convert into mol/mg.)

3. Supplements (Actual measurement data: Example MTX)
MTX)
3.1
3.1 HPLC peakFig.4 MTX 1 mM (500 nmol)

Fig.1 Buffer only

Supernatant A

Fig.2 Succinimide standard

Fig.5 MTX 0 nmol

curveSupernatant B

0 µM (DMF only)(without masking)Fig.3 Succinimide

Fig.6 TX 1 mM (500 nmol)

standard curve

Supernatant B

１00 µM

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Experiment Protocol 201
3.2
2 HPLC Quantitative Value

Con c. ofch em ical
co m poun d at
immo bilization(mM )259NH SSup A

259NH SSup B

M TX -NH2(n mol/5 00ul)

N HS supqu antit ative
co nc. withHPLC (uM )Succinim id evalu e o f 2 00u l(nm ol)Bac kgro un dremoval(nmol)

Immobil ized
M TX -NH2 at
1mg of beads(nmo l/m g)

COOH o r
NHS valu e(nm o l/mg)

Average(nmol/mg)

211.7

0079

15.9

0.0

0.0

201.5

0.1

5083

16.6

0.8

0.8

216.8

0.3

15099

19.9

4.0

4.0

212.7

1500

13627.2

11.3

11.3

215.9

01 0082 01.50.1

1 080

2 16.00.3

1 044

2 08.7

11 023

2 04.6200ul = 1mg of beads

3.3
3.3 Affinity purification by MTX immobilized beads
Salt Elution

Boi l Elution

KDa

KD a

M 0 0 .1 0. 3 1

2001169766

2 00116
9766

45

45

3131

21.5

21 .5

14. 4

14 .4

M 0 0.1 0 .3 1

M TX immo bilizat ion
co nc. (mM )DHFR

Condition
Beads
Amount of be ads
Binding, Washing buf fer
Elution buffer
Protein extract
Protein Conc.
Reaction time

: COOH be ads(S259PSSNENCOOH): 0.5mg: 150 mM KCl（200μl）: 1M KCl（30μl）: He La cytoplasmi c compartment: 3mg/ml（200μl）: 2h5

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Experiment Protocol 201
4. Notes
・Because micro-tube filters are not resistant to organic solvent, use them after diluting theorganic solvent with buffer.
・The value of supernatant A plus B shows the whole amount of COOH (NHS) of COOH (or NHSbeads) groups. When this value is significantly lower than 200 nmol/mg, it is considered that themasking was not performed correctly or NHS was separated by water contamination. In such acase, perform the immobilization again using new NHS beads.

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