U.S. Corporate Headquarters
400 Valley Rd.
Warrington, PA 18976
1(800) 523-2575 / (215) 343-6484
1(800)343-3291 fax
[email protected]
Polysciences Europe GmbH
Handelsstrasse 3
D-69214 Eppelheim, Germany
+(49) 6221-765767
+(49) 6221-764620 fax
[email protected]
Polysciences Asia-Pacific, Inc.
2F-1, 207 DunHua N. Rd.
Taipei, Taiwan 10595
(886) 2 8712 0600
(886) 2 8712 2677 fax
[email protected]
Page 1 of 3
PolyLink Amine Linker Kit with Columns and Reagents for Enzyme
Immobilization
INTRODUCTION
Polysciences offers the PolyLink Amine Linker Kit with Columns and Reagents for covalently coupling enzymes to
®
amino-functionalized amino acrylate beads. The kit includes 1 gram each of 4 different Purolite amine functionalized
resins along with all of the necessary Polysciences glutaraldehyde and buffer solution needed to carry out a standard
coupling protocol to bond your enzyme to the resins. The resins are individually packaged in ready-to-use 10 mL columns
with a bottom frit to allow rapidly treating the resins with reagents, filtering off excess reagents, and rinsing, all in one
vessel. The total working volume of the columns is 22 mL when the column head-space is included.
Figure 1: Activation of amino resin using glutaraldehyde and covalent immobilization of enzyme.
The included resins have 2 different linker lengths and 2 different porosities to allow you to rapidly identify the best resin
for immobilizing your enzyme. Amino resins can be used to covalently immobilize enzymes, following preactivation using
glutaraldehyde, as shown in Figure 1. Reaction of the aldehyde groups with amino groups of enzymes to form Schiff
bases is fast and gives stable multipoint covalent binding. If desired, the imine linkages can be made even more stable by
reducing the double bonds using a borohydride or another compatible reducing agent (reducing agent is not included in
this kit).
Table 1: Specifications of resins included in the kit.
Product
ID
50275A
50275B
50275C
50275D
Product Description
ECR8310F Amino C2 acrylate
ECR8319F Amino C2 acrylate
ECR8405F Amino C6 acrylate
ECR8417F Amino C6 acrylate
Functional group Immobilization Surface Area Pore diameter Water content
[m2/g]
[Å]
[%]
NH2 (short spacer)
Covalent
> 70
850 - 1200
58 - 62
NH2 (short spacer)
Covalent
> 50
1600 - 2000
62 - 66
NH2 (long spacer)
Covalent
> 40
450 - 850
50 - 55
NH2 (long spacer)
Covalent
> 50
1600 - 2200
61 - 65
All resins in this kit have a particle size of 150 – 300 micrometers. Corresponding resins with larger particle size (300 – 700
micrometers) are available as the M grade.
© Polysciences, Inc. Rev. #003
Active: 3 January 2013
Technical Data Sheet 978
U.S. Corporate Headquarters
400 Valley Rd.
Warrington, PA 18976
1(800) 523-2575 / (215) 343-6484
1(800)343-3291 fax
[email protected]
Polysciences Europe GmbH
Handelsstrasse 3
D-69214 Eppelheim, Germany
+(49) 6221-765767
+(49) 6221-764620 fax
[email protected]
Polysciences Asia-Pacific, Inc.
2F-1, 207 DunHua N. Rd.
Taipei, Taiwan 10595
(886) 2 8712 0600
(886) 2 8712 2677 fax
[email protected]
MATERIALS
Materials supplied in kit
• Column 1 (50275A): 1g wet ECR8310F Amino C2 acrylate
• Column 2 (50275B): 1g wet ECR8319F Amino C2 acrylate
• Column 3 (50275C): 1g wet ECR8417F Amino C6 acrylate
• Column 4 (50275D): 1g wet ECR8405F Amino C6 acrylate
• 4 end caps for the columns – to be used after the snap-tabs have been removed.
• 4 screw caps for the columns.
• Bottle 1 (Component A): Phosphate buffered saline (PBS), 2 x 225ml
• Bottle 2 (Component B): 8% Glutaraldehyde in PBS, empty labeled storage bottle
• 25% Glutaraldehyde, 2 x 10ml ampules
Materials not supplied
• Distilled water: 50ml
• Enzyme: sufficient enzymatic preparation to allow adding 50mg of protein for each gram of wet resin (200 mg total of
active enzyme will be required to treat all 4 columns in this kit). As with other parts of this procedure, this amount is an
approximate recommended amount. You may find that a different amount is more appropriate with your enzyme or for
your application.
• Solution of 0.5M NaCl in PBS (Component C): 20 mL
PROCEDURE
Researchers are advised to optimize the use of enzyme in any application, as the suggested procedures may not be
ideal. Other buffers and pHs that are optimal for specific enzymes can be used. Use of low concentration buffers is
typically recommended.
Procedure to prepare 8% glutaraldehyde in PBS solution (Component B)
1. Pipette 10ml of phosphate buffered saline (PBS) into Bottle 2.
2. Using ampoule cracker, open 10ml ampoule of 25% glutaraldehyde.
3. Pipette 5ml of 25% glutaraldehyde into Bottle 2.
4. Mix well. Store at 4˚C. Note: Glutaraldehyde can be unstable at a pH of 7.4 and may start to polymerize. Please inspect
the 8% glutaraldehyde/PBS solution prior to each use. If turbid or cloudy, discard and prepare a fresh solution.
Procedure to prepare 0.5 M NaCl in PBS (Component C)
5. Add 0.5844g NaCl to a clean vial.
6. Add 20 mL of PBS solution provided (Component A)
7. Mix at room temperature until all NaCl has dissolved. Procedure for Immobilization
a. Washing of the resin/Conditioning of the resin
8. Add 2ml of PBS into each supplied column containing resin and cap tightly.
9. Mix for 5 minutes at room temperature (20-25°C) on a rocker table, rotary shaker or any other kind of shaker that
provides end-to-end mixing. Avoid using a magnetic stirring since this can destroy the beads.
10. Filter and remove supernatant. If you have not done so already you will need to remove the snap-tab from the bottom
of the column to allow performing this filtration. After this has been done, the supplied cap should be used to seal the
bottom end of the column for all processes when liquid is present or when you want to prevent drying of the beads.
b. Pre-activation of the resin
11. Add 4ml of 8% glutaraldehyde in PBS (Component B) into each column and cap tightly.
12. Mix for 60 minutes at room temperature on a rocker table, rotary shaker or any other kind of shaker that provides end
to-end mixing. Avoid using a magnetic stirring since this can destroy the beads.
13. Filter the supernatant with vacuum.
14. Rinse the activated resin in each column with 2ml distilled water (4 times).
15. Remove supernatant from each rinse (vacuum max 600mmHg).
16. Wash the activated resin in each column with 2ml PBS (2 times).
17. Remove supernatant from each rinse.
c. Enzyme Immobilization
18. Prepare enzyme solution: Based on the known concentration or assay of your enzyme solution, add sufficient
enzyme solution to a vial so that a total of 200 mg of active enzyme has been added to the vial. Add sufficient PBS
© Polysciences, Inc. Rev. #003
Active: 3 January 2013
Technical Data Sheet 978
U.S. Corporate Headquarters
400 Valley Rd.
Warrington, PA 18976
1(800) 523-2575 / (215) 343-6484
1(800)343-3291 fax
[email protected]
Polysciences Europe GmbH
Handelsstrasse 3
D-69214 Eppelheim, Germany
+(49) 6221-765767
+(49) 6221-764620 fax
[email protected]
Polysciences Asia-Pacific, Inc.
2F-1, 207 DunHua N. Rd.
Taipei, Taiwan 10595
(886) 2 8712 0600
(886) 2 8712 2677 fax
[email protected]
buffer to this solution so that the total volume is 16 mL. Transfer 4 mL of this enzyme/ buffer solution into each of the four
columns.
19. Mix for 18 hours at room temperature on a rocker table, rotary shaker or any other kind of shaker that provides end-toend mixing. Avoid using a magnetic stirring since this can destroy the beads.
20. Stop mixing.
21. Filter and recover supernatant (vacuum max 600mmHg).
22. Rinse the resin in each column with 2ml of PBS (2 times).
23. Remove supernatant from each rinse (vacuum max 600mmHg).
24. Add 4 mL of the 0.5M NaCl solution in PBS (Component C) to the resin in each column and mix for 45min at room
temperature.
25. Rinse the resin in each column with 2ml of PBS (2 times).
26. Remove supernatant from each rinse (vacuum max 600mmHg).
27. You can determine enzyme immobilization yield by protein determination in the supernatant
28. Transfer the resin with immobilized enzyme to a storage container and store in the refrigerator at 4 ºC. If desired, the
resin with immobilized enzyme can also be stored in the columns after sealing both ends of the column using the caps
supplied.
STORAGE AND STABILITY
Store the components of the kit, columns containing resins, 25% glutaraldehyde ampoules and Bottle 2 at 4˚C. Do not
freeze columns containing resins and immobilised enzymes since this will cause bead breakage.
SAFETY
Glutaraldehyde is harmful if absorbed through the skin. Avoid contact with eyes, skin or clothing. Avoid breathing vapors.
Use only with adequate ventilation. Wear protective gloves and safety goggles. In case of contact, immediately flush eyes
or skin with plenty of water for at least 15 minutes. Remove contaminated clothing and shoes. Call a physician. Wash
contaminated clothing and shoes before wearing again. Please consult the Material Safety Data Sheet for more
information.
This product is for research use only and is not intended for use in humans or for in vitro diagnostic use.
Cat. #
Description
Size
Related Products for Enzyme Immobilization on Resins
50269-1
PolyLink Amine Linker Kit - F50 (50 g each of 4 resins with amine functionality)
50270-1
PolyLink Epoxy Linker Kit - F50 (50 g each of 3 resins with epoxy functionality)
50271-1
PolyLink Enzyme Adsorption Kit - F50 (50 g each of 5 resins for enzyme adsorption)
50272-­‐1 PolyLink Lipase Linker Kit -­‐ F50 (50 g each of 4 resins for lipase immobilization) 50273-1
PolyLink Enzyme Linker Kit - F50 (50 g each of 5 resins with various functionality for enzyme
immobilization) Related Products for Enzyme Immobilization on Microparticles
19540-1
Glutaraldehyde Kit for Amine and Blue Dyed Microspheres
1 kit 23964-1
Glutaraldehyde Kit with Hollow Fiber Filtering System
1 kit 16586-5
Polybead® Amino Microspheres, 0.10µm
5ml 15699-5
Polybead® Amino Microspheres, 0.20µm
5ml 07763-5
Polybead® Amino Microspheres, 0.50µm
5ml 17144-5
Polybead® Amino Microspheres, 0.75µm
5ml 17010-5
Polybead® Amino Microspheres, 1.00µm
5ml
17145-5
Polybead® Amino Microspheres, 3.00µm
5ml
19118-5
Polybead® Amino Microspheres, 6.00µm
5ml
Should any of our materials fail to perform to our specifications, we will be pleased to provide replacements or return the purchase price. We solicit your inquiries concerning
all needs for life sciences work. The information given in this bulletin is to the best of our knowledge accurate, but no warranty is expressed or implied. It is the user’s
responsibility to determine the suitability for their own use of the products described herein, and since conditions of use are beyond our control, we disclaim all liability with
respect to the use of any material supplied by us. Nothing contained herein shall be construed as a recommendation to use any product or to practice any process in violation
of any law or any government regulation.
© Polysciences, Inc. Rev. #003
Active: 3 January 2013
Technical Data Sheet 978