Product Information
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Carboxyl Magnetic Beads is magnetic bead with surface functional group -COOH. The magnetic bead consists of a single-crystal Fe3O4 sphere core and dextran coating layer. Through chemical modification of dextran, the carboxyl groups (-COOH) 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.
Through activation of Carboxyl Magnetic Beads with EDC, the ligands could be conjugated to the magnetic beads through primary amine groups such as antibody, protein, or peptide.
Specifications
| Core | Iron Oxide (Fe3O4) |
| Layer | Dextran |
| Functional group | carboxyl group (-COOH) |
| Mean Diameter of Particles | ~1 μm |
| 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 |
Material supplied
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Beads-Carboxyl provides Fe3O4 beads coated with dextran of an average ~1 μm in diameter. Carboxyl group, more than 0.2 mM, is coupled covalently to dextran. Beads-Carboxyl is supplied in phosphate buffered saline, pH 7.4, 0.02% Tween-20 and 0.09% NaN3
Additional material required
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•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
•Magnetic 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
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Preparation of Beads-Carboxyl for Use
1. Resuspend the Beads-Carboxyl thoroughly by pipetting or vortexing the vial.
2. Transfer an adequate amount of Beads-Carboxyl 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.
Activation of Beads-Carboxyl
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. Incubate with tilt rotation for 15 minutes at room temperature.
13. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
14. Discard the supernatant, then remove the tube from the magnetic stand.
Conjugation of Protein or Ligands
15. Add 50 μL MES Buffer containing 6-150 μg antibody or ligand, then resuspend the beads by pipetting.
16. Incubate with tilt rotation at room temperature for 90 minutes or at 4°C overnight.
17. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
18. Discard (or collect, if desired) the supernatant as unbound substances, then remove the tube from the magnetic stand.
19. Add 100 μL MES Buffer and resuspend the beads by pipetting.
20. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
21. Discard the supernatant, then remove the tube from the magnetic stand.
Stop the Reaction
22. Add 500 μL Quench Buffer and resuspend the beads by pipetting.
23. Incubate with tilt rotation for 30 minutes at room temperature.
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 Quench 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. Add 500 μL PBS, pH 7.4 (or preferred buffer) and resuspend the beads by pipetting.
30. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
31. Discard the supernatant, then remove the tube from the magnetic stand.
32. Repeat steps 30-31 twice.
33. Add 100 μL PBS, pH 7.4 (or preferred buffer) and resuspend the beads by pipetting.
34. Store the beads at 2-8°C.
Troubleshooting
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