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Activation Protocols for Biodyne® C and LoProdyne® LP Membranes |
Introduction
Certain procedures require that molecules be covalently immobilized on membranes. Typical applications requiring covalently immobilized ligands are immuno-capture assays, ELISAs, immobilization of amino-terminated oligonucleotides, gene probe tests, etc.
Several patented activation protocols for the covalent attachment of ligands to Biodyne C and LoProdyne LP membranes are provided to assist you. The procedures for Biodyne C membrane utilize cyanuric chloride, dicyclohexyl carbodiimide or glutaraldehyde. Carbonyl diimidazole is used for the activation of LoProdyne LP membrane. Please note that the procedures which use methylene chloride as a solvent must be performed beneath a fume hood. Also, membranes activated in methylene chloride should be protected against exposure to water or water vapor until use. In addition, glutaraldehyde can be added to the membranes in water or buffer (PBS).
If you need a pre-activated membrane offering a high density of covalent binding sites, please evaluate Immunodyne® ABC or UltraBind® membrane. These membranes are pre-activated solid phase supports for covalent immobilization of polyclonal and monoclonal antibodies, enzymes, purified or genetically engineered peptide antigens and amino-terminated oligonucleotides.
Concerning these activation protocols, you should be aware of our U.S. Patents 4,693,985 and 4,886,836. The purchase of Pall membrane provides you with a license under these patents. However, if media are purchased from a third party, no license is provided under Pall's patents. You should also note that while we are not aware of any patents of others relating to these protocols, we cannot make any representations concerning patents of third parties.
Several patented activation protocols for the covalent attachment of ligands to Biodyne C and LoProdyne LP membranes are provided to assist you. The procedures for Biodyne C membrane utilize cyanuric chloride, dicyclohexyl carbodiimide or glutaraldehyde. Carbonyl diimidazole is used for the activation of LoProdyne LP membrane. Please note that the procedures which use methylene chloride as a solvent must be performed beneath a fume hood. Also, membranes activated in methylene chloride should be protected against exposure to water or water vapor until use. In addition, glutaraldehyde can be added to the membranes in water or buffer (PBS).
If you need a pre-activated membrane offering a high density of covalent binding sites, please evaluate Immunodyne® ABC or UltraBind® membrane. These membranes are pre-activated solid phase supports for covalent immobilization of polyclonal and monoclonal antibodies, enzymes, purified or genetically engineered peptide antigens and amino-terminated oligonucleotides.
Concerning these activation protocols, you should be aware of our U.S. Patents 4,693,985 and 4,886,836. The purchase of Pall membrane provides you with a license under these patents. However, if media are purchased from a third party, no license is provided under Pall's patents. You should also note that while we are not aware of any patents of others relating to these protocols, we cannot make any representations concerning patents of third parties.
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Activation Protocols: Biodyne C Membrane
All incubation and wash steps to be performed in glass containers or trays. All steps, including weighing, to be performed in hood.
Materials:
- Cyanuric Chloride (TsT)
- Methylene Chloride (MeCl2)
- Triethylamine (TEA)
- Dicyclohexylcarbodiimide (DCCD)
- Phosphate Buffered Saline (PBS)
- Glutaraldehyde
- Biodyne C Membrane
TsT (Trichloro-s-Triazine or Cyanuric Chloride)
- Cut pieces of Biodyne C membrane.
- Soak membrane in 4.3 mL TEA and 65 mL of MeCl2 for 2 minutes.
- Add 2g TsT, agitate gently and then incubate for 15 minutes at room temperature.
- Wash membrane 4X, 5 minutes per wash, with 50 mL MeCl2.
- Air dry 2 minutes; place in vacuum desiccator until use.
DCCD (Dicyclohexylcarbodiimide)
- Soak membrane in 50 mL of MeCl2 containing 5g (10% w/v) DCCD for 30 minutes.
- Wash membrane 4X, 5 minutes per wash, with 50 mL MeCl2.
- Air dry 2 minutes; place in vacuum desiccator until use.
Glutaraldehyde
- Cut 13 mm discs of sample media.
- Soak discs in 50 mL of PBS containing 0.5% - 5% (2.5 mL) glutaraldehyde for 2 hours.
- Wash membranes 4X, 5 minutes per wash, with 50 mL PBS.
- Air dry 10 minutes and place in vacuum desiccator or store in PBS until use.
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Activation Protocol: LoProdyne LP Membrane
LoProdyne LP membrane has hydroxyl surface chemistry. The membrane binds very little protein in standard binding tests using IgG or BSA. The membrane can be activated for covalent attachment using N, N® carbonyldiimidazole (CDI) in methylene chloride as follows:
- Dissolve 0.49 g CDI in 45 mL MeCl2.
- Add to a glass dish under a fume hood.
- Immerse sheet of LoProdyne LP membrane in this solution for 15 minutes, RT.
- Wash membrane 4X with 40 mL per wash MeCl2, 5 minutes per wash.
- Air dry at 60 °C for 3 minutes.
- Store in vacuum desiccator until use.
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