Corporate Headquarters Europe – Germany Asia -­‐ Taiwan 400 Valley Road Polysciences Europe GmbH Polysciences Asia-­‐Pacific, Inc. Warrington, PA 18976 Handelsstr. 3 2F-­‐1, 207 Tunhwa N. Rd. 1-­‐800-­‐523-­‐2575 D-­‐69214 Eppelheim, Germany Taipei, Taiwan 10595 FAX 1-­‐800-­‐343-­‐3291 (49) 6221-­‐765767 (886) 2 8712 0600 Email: [email protected] FAX (49) 6221-­‐764620 FAX (886) 2 8712 2677 www.polysciences.com Email: [email protected] Email: [email protected] TECHNICAL DATA SHEET #992
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PolyLink Epoxy Resins Kit with Columns and Reagents for Enzyme
Immobilization
Introduction
Polysciences offers the PolyLink Epoxy Resins Kit with Columns and Reagents for covalently coupling enzymes to
epoxy-functionalized Methacrylate and acrylic/styrene beads. The kit includes 1 gram each of 3 different Purolite® epoxy
functionalized resins along with all of the necessary Polysciences buffer solutions 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: Covalent immobilization of enzyme on epoxy functionalized resin.
H2N
OH
H2N
O
O
O
NH
O
NH
H2N
O
O
Enzyme
OH
Cov alently immobilized enzyme
Purolite ECR Epoxy resin
The included resins have different porosities, polymer backbone with differing hydrophilic properties to allow you to rapidly
identify the best resin for immobilizing the enzyme of choice. Epoxy resins can be used to covalently immobilize enzymes
without any pretreatment, as shown in Figure 1. The reaction of the epoxy groups with amino groups of enzymes is
relatively fast and gives stable multipoint covalent binding.
Table 1: Specifications of resins included in the kit.
Product
ID
50279A
50279B
50279C
Product Description
ECR8205F Epoxy
Methacrylate
ECR8214F Epoxy
Methacrylate
ECR4204F Epoxy
acrylic/styrene
Functional group Immobilization Surface Area Pore diameter Water content
[m2/g]
[Å]
[%]
Epoxy
Covalent
> 80
450-600
52 - 57
Epoxy
Covalent
> 60
1200 - 1800
60 - 66
Epoxy
Covalent
> 140
275 - 450
41 - 46
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.
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Corporate Headquarters Europe – Germany Asia -­‐ Taiwan 400 Valley Road Polysciences Europe GmbH Polysciences Asia-­‐Pacific, Inc. Warrington, PA 18976 Handelsstr. 3 2F-­‐1, 207 Tunhwa N. Rd. 1-­‐800-­‐523-­‐2575 D-­‐69214 Eppelheim, Germany Taipei, Taiwan 10595 FAX 1-­‐800-­‐343-­‐3291 (49) 6221-­‐765767 (886) 2 8712 0600 Email: [email protected] FAX (49) 6221-­‐764620 FAX (886) 2 8712 2677 www.polysciences.com Email: [email protected] Email: [email protected] Materials
Materials supplied in kit
• Column 1 (50279A): 1g wet ECR8205F Epoxy Methacrylate
• Column 2 (50279B): 1g wet ECR8214F Epoxy Methacrylate
• Column 3 (50279C): 1g wet ECR4204F Epoxy acrylic/styrene
• 3 end caps for the columns – to be used after the snap-tabs have been removed.
• 3 screw caps for the columns.
• Bottle 1 (Component A): Phosphate buffer solution pH 8, 1.0 Molar; 200 mL
• Bottle 2 (Component B): Phosphate buffered saline (PBS), 225ml
Materials not supplied
• Distilled water
• Enzyme: sufficient enzymatic preparation to allow adding 50mg of protein for each gram of wet resin (150 mg total of
active enzyme will be required to treat all 3 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.
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 high concentration buffers is
typically recommended for the immobilization process. Please note the optimum pH stability of the epoxy resins is in the
range 5 – 9.
Procedure for Immobilization
a. Washing of the resin/Conditioning of the resin
1. Add 2ml of distilled water into each supplied column containing resin and cap tightly.
2. 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.
3. 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.
4. Add 2ml of buffer (Component A) into each supplied column containing resin and cap tightly.
5. 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.
6. 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. Enzyme Immobilization
7. 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 150 mg of active enzyme has been added to the vial. Add sufficient buffer
(Component A) to this solution so that the total volume is 12 mL. Transfer 4 mL of this enzyme/ buffer solution into
each of the four columns.
8. Mix for 18 hours 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.
9. Stop mixing.
10. Leave the resin and enzyme mix to react without agitation for approximately 20 hours at temperature 20-25°C
11. Filter and recover supernatant (vacuum max 600mmHg).
12. Rinse the resin in each column with 2ml of PBS Component B (2 times).
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Corporate Headquarters Europe – Germany Asia -­‐ Taiwan 400 Valley Road Polysciences Europe GmbH Polysciences Asia-­‐Pacific, Inc. Warrington, PA 18976 Handelsstr. 3 2F-­‐1, 207 Tunhwa N. Rd. 1-­‐800-­‐523-­‐2575 D-­‐69214 Eppelheim, Germany Taipei, Taiwan 10595 FAX 1-­‐800-­‐343-­‐3291 (49) 6221-­‐765767 (886) 2 8712 0600 Email: [email protected] FAX (49) 6221-­‐764620 FAX (886) 2 8712 2677 www.polysciences.com Email: [email protected] Email: [email protected] 13. Remove supernatant from each rinse (vacuum max 600mmHg).
14. Add 4 mL of the PBS Component B to the resin in each column and mix for 45min at room temperature.
15. Rinse the resin in each column with 2ml of PBS Component B (2 times).
16. Remove supernatant from each rinse (vacuum max 600mmHg).
17. You can determine enzyme immobilization yield by protein determination in the supernatant
18. 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 and columns containing resins at 4˚C. Do not freeze columns containing resins and
immobilised enzymes since this will cause bead breakage.
Safety
This product is for research use only and is not intended for use in humans or for in vitro diagnostic use.
TO ORDER
In The U.S. Call: 1(800) 523-2575 • (215) 343-6484
In The U.S. Fax: 1(800) 343-3291 • (215) 343-0214
In Germany Call: +(49) 6221-765767
In Germany Fax: +(49) 6221-764620
In Asia Call: (886) 2 8712 0600
In Asia Fax: (886) 2 8712 2677
Order online anytime at www.polysciences.com
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