Pyridylamination Manual Kit
Cat. #4480
v1001
Table of Content
I. Description................................................................................................................. 2
II. Kit component......................................................................................................... 3
III. Storage....................................................................................................................... 3
IV. Reagents required................................................................................................. 3
V. Protocol
A. Pyridylamination......................................................................................... 3
B. Purification of PA-sugar chain................................................................ 4
VI. Control experiment.............................................................................................. 5
VII. Reference................................................................................................................ 6
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1
Cat. #4480
Pyridylamination Manual Kit
v1001
I. Description :
This kit is designed to enable fluorescence labeling of sugar chain with 2-aminopyridine without a special instrument. To the reduced termini residue of sugar chain,
2-aminopyridine is bound with reduced amino reaction. Then, sugar chain is induced
into a stable fluorescent labeled pyridylamino (PA)-sugar chain (see figure below).
After pyridylamination, PA-sugar chains are recovered by removing excess reagents
through column chromatography with Cellulose Cartridge. The recovery efficiency
with Cellulose Cartridge is as good as with evaporation method using an instrument
for automated pyridylamination. As the PA-sugar chain prepared with this kit includes
fluorescence residue, highly sensitive analysis with HPLC is achieved.
Note :
The pyridylamination efficiency and recovery efficiency from Cellulose Cartridge differs depending on a structure of sugar chain. This kit is optimized for double stranded
complex N-Glycan (Biantennary sugar chain), and it is supplied as control in this product ＊ . When structure of target sugar chain, especially types of sugar chain residue at
reduced termini and molecular weight of sugar chain are different from the control, it
is recommended to optimize the condition through preliminary experiment.
This kit is not suitable for the labeling of monosaccharide.
<Pyridylamination of Sugar chain>
O
...... O
O
NHAc
NHAc
N
AcOH
O
O
NHAc
Reduction
CH=N
N
NHAc
（CH3）
2NH・BH3
O
...... O
H, OH
H2N
Formation of
schiff base
...... O
O
O
NHAc
CH2-NH2
N
NHAc
＊ : Structure of Biantennary sugar chain, which is supplied with this kit
Galβ1-4GlcNAcβ1-2Manα1
Galβ1-4GlcNAcβ1-2Manα1
2
6
Manβ1-4GlcNAcβ1-4GlcNAc
3
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Pyridylamination Manual Kit
Cat. #4480
v1001
II. Kit Components (20 reactions including control reaction) :
[Package 1]
(1) 2-Aminopyridine
2×300 mg
(2) Borane-dimethylamine complex
2×100 mg
(3) Acetic acid
3 ml
(4) Biantennary sugar chain
500 pmol/50 μl
(5) PA-Biantennary sugar chain
100 pmol/100 μl
[Package 2]
(6) Cellulose Cartridge Column
(7) Adaptor
(8) Syringe
III. Storage :
20×0.5 ml
1 piece
3×10 ml
Package 1 ((1) - (5)) : -20℃
Package 2 ((6) - (8)) : Room temperature
＊ 2 years from date of receipt under proper storage conditions.
IV. Reagents required (not supplied in the kit) :
Butanol (Purity : high grade)
Ethanol (Purity : high grade)
Acetic acid (Purity : high grade)
Ammonium bicarbonate (75 mM aqueous solution)
Sterilized distilled water
approx. 500 ml
approx. 500 ml
approx. 3 ml
approx. 500 ml
V. Protocol :
A : Pyridylamination
[Preparation]
Set air-oven or water bath at 80℃ . (Heat block can be used for this, however temperature does not stabilize, thus does not perform greatly).
[Mixing reagents]
- Coupling reagent
Add 100 μl of (3) Acetic acid into vial of (1) 2-Aminopyridine (300 mg). Occasionally, heat at 80℃ , and evenly dissolve the agents by applying vortex. Final
volume of reagents will be about 250 μl.
(Note 1)One vial of (1) 2-Aminopyridine is for ten reactions. After preparing
reagents, store in plastic tube with tight cap at -20℃ . The prepared
reagent should be used within three month.
(Note 2)Use care for storing the coupling reagent, it might be coagulated below room temperature. Also the reagents are very viscous, so it will
mix easily when heated reasonably.
- Reducing agent
Weigh 20 mg of (2) Borane-dimethylamine complex in plastic tube and add
100 μl of (3) Acetic acid then dissolve well to make reducing agents.
(Note 3)(2) Borane-dimethylamine complex is hygroscopic, thus reagent vial
must be replaced in room temperature before weighing them and
must be weighted in low humidity place. Weighing process must be
done swiftly. After the use, bottle must be caped tightly and cover
with Parafilm.
(Note 4)Reducing agent is prepared for each experiment. Use them the day
when it was prepared.
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Pyridylamination Manual Kit
Cat. #4480
v1001
[Reaction]
1. Place sugar chain sample (50 pmol-50 nmol) into 1.5 ml plastic tube with screw
cap. Dry the sample sufficiently by freeze dry or concentrated centrifuge.
(Note 5)Use sugar chain sample without salt, protein, or lipid as much as possible. Generally, for purification of sugar chain before labeling, Cellulose
Cartridge Glycan Preparation kit (Cat. #4403) or column chromatography such as gel filtration is used.
2. Add 20 μl of coupling reagent to sample, mixed well. Incubate at 80℃ for
1.5 hours.
(Note 6)When water bath is used for heating, the cap must be covered with
Parafilm or Teflon seal tape to avoid water to get in to the reaction
mixture. Also, after the reaction is completed, wipe off the water on
the out side of the bottle, then open the cap.
3. Add 20 μl of reducing agent to the reaction mixture and mix well. Then, incubate at 80℃ for 1 hour.
4. Immediately purify PA-sugar chain after the reaction.
(Note 7)When purification is not done immediately, store the reaction mixture
at below -20℃ . Purification must be done within a week.
B : Purification of PA-sugar chain (operation must be done at the room temperature (2025℃ )).
[Preparation]
Preparation of the solvent for PA-sugar chain purification
Solvent 1 : Butanol/Ethanol/Water/Acetic acid 4 : 1 : 0.97 : 0.03 (volume ratio)
Solvent 2 : Ethanol/75 mM Ammonium Bicarbonate 1 : 2 (volume ratio)
(Note 1)About 25 ml of solvent 1 and about 15 ml of solvent 2 are necessary
for a purification.
(Note 2)Solvent must be prepared each time. When storage is required, store
in a container that can be well sealed in cool place. Use within a week.
[Purification]
1. Pre-treatment of cartridge
a) Attach adaptor to cartridge
b) Inject 10 ml of sterilized distilled water into syringe, and then attach adaptor
to wash the cartridge. (Send fluid 2-5 drops per seconds.) Repeat this at least
three times. Finally, send air through syringe to remove all water out of the
cartridge.
c) Inject 10 ml of Solvent 2 into syringe, and then attach adaptor to wash cartridge. (Send fluid 2-5 drops per seconds.) Finally, send air through syringe
to remove all solvent out of the cartridge.
d) Inject 10 ml of Solvent 1 into syringe, and attach adaptor to equilibrate cartridge. (Send fluid 2-5 drops per seconds.) Fill and leave slight Solvent 1 in
the top of the column bed.
2. Preparation for sample reagent
a) Add 1 ml of Solvent 1 to reaction mixture (40 μl), and stir well. Do not centrifuge reaction mixture.
(Note 3)Protein in reaction mixture may precipitate, however, do not centrifuge and apply suspension solution to column as it is.
(Note 4)When mixed sample reagents were clearly separated into two layers, by applying Solvent 1 to the sample reagents, it might dilute
and even out. However, when addition of 5 ml of Solvent 1 did not
make any difference, the concentration of the contaminant other
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URL:http://www.takara-bio.com
Pyridylamination Manual Kit
Cat. #4480
v1001
than sugar chain, such as salt is rather high. And it is possible that
pyridylamination is not done appropriately. In such case, after dry
and harden the sample reagents again, remove salt by gel filtration. Then, try pyridylaminating it again.
3. Applying sample reagent
a) Mix sample reagents well. Then inject it to cartridge cylinder without adaptor.
b) Attach adaptor, and syringe which injected air, then slowly push out sample
reagents with syringe’ s air. (1 drop per second). Stop this just before sample reagent is at the top of the column bed. Pay attention to cartridge, so it
would not flush out.
(Note 5)For confirmation, do not throw out eluate from column until result
analysis is done. Collect then in plastic tube. Do the same for cleaning solution below in 4 : washing cartridge.
c) Wash reaction tube with 1 ml of Solvent 1. Inject it to cartridge cylinder, then
through the column as described above section b.
4. Washing cartridge
Inject 10 ml of Solvent 1 to syringe, then attach adaptor to clean cartridge.
Cartridge should not be flush out in the process. (One drop per second). In the
end, push 2 ml of air out of syringe to all the solvent in the cartridge will be out.
(Note 6)When the volume of sample reagent is more than average volume, adjust the volume of the total volume of sample reagent and
washing solvent (Solution 1) must be 12 ml.
5. Elution of sugar chain
a) Remove adaptor. Then, inject 2 ml of Solvent 2 to cartridge.
b) Attach adaptor, and syringe which injected air, then slowly push out sample
reagents with syringe's air. (1 drop per second). Collect eluate in 1.5 ml plastic tube (divide into two of 1 ml) or 5 ml test tube. Finally, push out all the
solvent in the cartridge using air.
c) Use centrifuge concentrator to desiccate the eluate. Then offer to HPLC
analysis.
(Note 7) This cartridge is disposable.
VI. Control experiment :
As it described above, process Pyridylamination and Purification of PA-sugar chain by
using10 μl (100 pmol) of (4) Biantennary Sugar Chain. Analyze PA-sugar chain with
HPLC. Compare Pyridylamination rate of this with (5) PA-Biantennary Sugar Chain.
(Note) This kit contains 20 reactions including control reaction. Please not, when
control experiments were used, amount of sample reaction will be reduced.
- Using Pyridylamination Manual Kit for HPLC analysis of PA-sugar chain
[Procedure]
Dry Biantennary Sugar Chain, which have done pyridylamination with this kit. Then
add 100 μl of distilled water to dissolve. Take 5 μl of this and add 5 μl (5 pmol) of
standard material PA-Sugar Chain 003 (Cat. #4103). Then analyze with HPLC.
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Cat. #4480
Pyridylamination Manual Kit
v1001
[Result]
50000
Fliorescence
40000
A
30000
B
20000
10000
0
0
20
40
60
Time (min)
A : PA-Biantennary sugar chain (pyridylamination of Biantennary Sugar Chain by
using this kit)
B : Standard Material PA-Sugar Chain 003 (Cat. #4103) ＊
＊ : This is not included in this kit
HPLC Condition
Cloumn : TSKgel Amide-80 (4.6×250) (TOSOH Corporation)
Solvent A : 500 mM Acetic acid-Triethylamine (pH7.3)/CH3CN/H2O (10/75/15)
Solvent B : 500 mM Acetic acid-Triethylamine (pH7.3)/CH3CN/H2O (10/50/40)
Gradient : 0％ B for 15 min, and then 0-100％ B/50 min
Flow rate : 1 ml/min
Column temperature : 40℃
Fluorescence : Ex. (310 nm), Em. (380 nm)
VII. Reference :
1) Kondo, A., et al .(1990) Agric. Biol. Chem . 54, 2169.
2) Ohara, K., et al . (1991) J. Chromatgr . 586, 35.
NOTICE TO PURCHASER : LIMITED LICENSE
[M14] Cellulose Cartridge
This product is covered by the claims of Japanese Patent No. 3885912.
NOTE : This product is intended to be used for research purpose only. They are not to be used for
drug or diagnostic purposes, nor are they intended for human use. They shall not to be
used products as food, cosmetics, or utensils, etc.
Takara products may not be resold or transfered, modified for resale or transfer, or used to
manufacture commercial products without written approval from TAKARA BIO INC.
If you require licenses for other use, please call at +81 77 543 7247 or contact from our
website at www.takara-bio.com .
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Phone : +81-77-543-7247 Fax : +81-77-543-9254