Product Information
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NTA-Ni Magnetic Beads is designed for rapid purification of 6xHis-tagged proteins. NTA-Ni Magnetic Beads has nitrilo- triacetic acid (NTA) groups with charged nickel covalently bind to surface dextran of Beads. Due to the high affinity, NTA-Ni Magnetic Beads can be used for capturing 6xHis-tagged proteins. Bound 6xHis-tagged proteins can be temporarily immobilized under magnetic attraction, so the other parts in supernatant can be removed easily and efficiently. Bound proteins can be directly used in downstream applications or be eluted off the beads. The capacity of purified 6xHis-tagged proteins (~35kDa) captured by NTA-Ni Magnetic Beads is approximately 5 mg/mL.
Binding characteristics
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Specifications
| Core | Iron Oxide (Fe3O4) |
| Layer | Dextran |
| Surface protein | NTA-Ni |
| Mean Diameter of Particles | ~1 μm |
| Storage Buffer | 20% ethanol |
| Storage temp. | 2 ~ 8 ℃ |
| Capacity | 5 mg/mL |
Material supplied
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Beads-NTA-Ni contains coated magnetic beads of an average ~1 μm in diameter. The beads are suspended in 20% ethanol reagent.
Additional material required
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• Beads-NTA-Ni Binding/Wash Buffer
- 50 mM sodium phosphate, pH 7.4
- 300 mM NaCl
- 0.02 % Tween 20
• Magnetic stand: Magstand for the best performance
• Tilt rotation device
• Beads-NTA-Ni Elution Buffer
- 50 mM sodium phosphate, pH 7.0
- 300 mM NaCl
- 500 mM Imidazole
- 0.1 % Tween 20
• Eppendorf tubes & pipettes
Protocol
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Preparation of Beads-NTA-Ni for use
1. Resuspend the Beads-NTA-Ni thoroughly by pipetting or vortexing the vial.
2. Transfer 100 μL Beads-NTA-Ni suspension into a clean Eppendorf tube.
3. Add 900 μL Binding/Wash buffer and resuspend the beads by pipetting.
4. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
5. Discard the supernatant by aspiration with a pipette.
6. Remove the tube from the magnetic stand.
7. Add 1 mL Binding/Wash buffer and resuspend the beads by pipetting.
8. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
9. Discard the supernatant, then remove the tube from the magnetic stand.
10. Repeat steps 7-9 twice.
11. The beads are now ready for purification of 6xHis-tagged proteins.
NOTE: Wash the Beads-NTA-Ni at least three times before use.
Purification of 6xHis-tagged Proteins
12. Mix 100 μL clear lysate sample and 900 μL Binding/Wash Buffer with the beads thoroughly by pipetting.
13. Incubate with tilt rotation for 30 minutes at room temperature.
14. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
15. Collect (or discard) the supernatant as unbound substances by aspiration with a pipette, then remove the tube from the magnetic stand.
16. Add 1 mL Binding/Wash 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. Collect (or discard) the supernatant as unbound substances, then remove the tube from the magnetic stand.
19. Repeat steps 16-18 two more times.
20. Proceed to elution of 6xHis-tagged proteins.
Elution of 6xHis-tagged Proteins
21. Add 500 μL Elution Buffer and gently resuspend the Beads-NTA-Ni-(6xHis-tagged proteins) complex by vortexing and pipetting.
22. Incubate with tilt rotation for 15 minutes at room temperature.
23. Place the tube on the magnetic stand for 30-60 seconds and collect the supernatant into a clean tube.
24. If required, repeat steps 21-23.
NOTE: The first eluate (from step 22) contains the majority of the purified 6xHis-tagged proteins. If required, both eluates (from steps 22 & 23) can be combined.
Troubleshooting
| Troubles | Solutions |
| The yield of protein is low. | 1. Check collecting supernatant from some steps (Step 14, 17, and 22) by SDS-PAGE. 2. Incubation time and temperature can be optimized depending on each sample. 3. We strongly recommend using 500 mM imidazole in the elution buffer. 4. Check the pH and composition of all buffers and solutions. 5. Make sure the beads are suspended thoroughly during both the binding and elution steps. |
| Proteins degrade during purification. | 1. Carry out the purification procedures at low temperature (e.g., 2–8°C). 2. Use proper protease inhibitors in lysis, binding/wash, and elution buffer. |
| The beads adhere on the tip or tube. | 1. Decrease the salt concentration (e.g., 50 mM NaCl) in the binding/wash buffer. 2. Increase Tween 20 concentration (e.g., 0.1%) in the binding/wash buffer. |
| Beads are hard to immobilize using the magnet stand. | 1. Make sure the tube is in direct contact with the magnet stand. 2. Use the Magstand magnetic stand for the best performance. |
| Purified His-tagged protein can’t be quantified using standard methods such as Bradford or BCA. | 1. The imidazole in the elution buffer may interfere with these assays. Either dialyze the sample or dilute it to the optimal imidazole concentration for the protein quantification reagent used. 2. Check that the beads are not in suspension. The remaining beads may interfere. |