Product Information
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Amine Magnetic Beads is magnetic bead with surface functional group -NH2. The magnetic beads consist of Fe3O4 magnetic sphere core and being coated with dextran. Through chemical modification of dextran, the primary amino group (-NH2) are joined to the magnetic beads through a short hydrophilic linker. The hydrophilic surface ensures the magnetic beads excellent dispersion ability and easy handling property in a wide variety of buffers.
The magnetic beads with surface-reactive amino groups allow immobilization of ligands such as proteins, peptides, carbohydrates or other target specific molecules.
Specifications
| Core | Iron Oxide (Fe3O4) |
| Layer | Dextran |
| Functional group | primary amino group (-NH2) |
| Mean Diameter of Particles | ~1 um |
| Storage Buffer | PBS pH-7.4 with 0.09% Sodium Azide and 0.02% Tween-20 |
| Storage temp. | 2 ~ 8 ℃ |
| Ligand density | 0.2 mM |
Additional material required
-
• MES Buffer (pH 6.0):
100 mM MES and 500 mM NaCl
• PBS, pH 7.4:
137 mM NaCl, 8.1 mM Na2HPO4,
1.47 mM KH2PO4 and 2.7 mM KCl
• Quench Buffer:
TBS,
pH 8.0 or 5-10 mM hydroxylamine
• Desired antibody or ligand
• Magntic stand: Magstand for the best performance
• EDC [1-(3-dimethylaminopropyl)-3-ethylcarbo- diimide hydrochloride], C8H17N3·HCl, MW = 191.7, CAS No. 25952-53-8
• MES [2-(morpholino) ethanesulfonic acid], C6H13NO4S·H2O, MW = 213.25, CAS No.145224-94-8
• NHS [N-hydroxysuccinimide], C4H5NO3, MW = 115.09, CAS No. 6066- 82-6
• Tilt rotation device or vortexer
• Eppendorf tubes & pipet
Protocol
-
Preparation of Beads-Amine for Use
1. Resuspend the Beads-Amine thoroughly by pipetting or vortexing the vial.
2. Transfer 100 μL Beads-Amine into a clean tube.
3. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
4. Discard the supernatant by aspiration with a pipette.
5. Remove the tube from the magnetic stand.
6. Add 200 μL MES Buffer and resuspend the beads by pipetting.
7. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
8. Discard the supernatant, then remove the tube from the magnetic stand.
9. Repeat steps 6-8 twice.
Conjugation of Protein or Ligands
10. Prepare 50 mg/mL EDC solution in MES Buffer and 50 mg/mL NHS solution in MES Buffer separately.
NOTE: Both EDC solution and NHS solution should be freshly prepared, protected from light, and kept on ice before use.
11. Add 60 μL MES Buffer, 20 μL EDC solution, and 20 μL NHS solution to the tube from step 9, then resuspend the beads by pipetting.
12. Add 50 μL MES Buffer containing 6-150 μg antibody or ligand, then resuspend the beads by pipetting.
13. Incubate with tilt rotation at room temperature for 90 minutes or at 4°C overnight.
14. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
15. Discard (or collect, if desired) the supernatant as unbound substances, then remove the tube from the magnetic stand.
16. Add 100 μL MES Buffer and resuspend the beads by pipetting.
17. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
18. Discard the supernatant, then remove the tube from the magnetic stand.
Stop the Reaction
19. Add 500 μL Quench Buffer and resuspend the beads by pipetting.
20. Incubate with tilt rotation for 30 minutes at room temperature.
21. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
22. Discard the supernatant, then remove the tube from the magnetic stand.
23. Add 500 μL Quench Buffer and resuspend the beads by pipetting.
24. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
25. Discard the supernatant, then remove the tube from the magnetic stand.
26. Add 500 μL PBS, pH 7.4 (or preferred buffer) and resuspend the beads by pipetting.
27. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
28. Discard the supernatant, then remove the tube from the magnetic stand.
29. Repeat steps 26-28 twice.
30. Add 100 μL PBS, pH 7.4 (or preferred buffer) and resuspend the beads by pipetting.
31. Store the beads at 2-8°C.