Product Information
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Beads-Protein A is designed as a rapid and simple tool or immunoprecipitation, purification/ depletion assays, and other magnetic separation applications. Antibody can easily bind to the Beads due to its’ high affinity with protein A. Via the antibody specific binding ability, the target protein along with Beads-Protein A could be temporarily immobilized at tube wall, so the other parts in the supernatant can be removed easily and efficiently under magnetic attraction.
Binding characteristics
The binding strength of Beads-Protein A to different immunoglobulins is listed as below:
Table 1. Binding strength of Beads-Protein A
| Species | Binding strength |
|---|---|
| Human IgG (normal) | ＋＋＋＋ |
| Mouse IgG1 | ＋ |
| Rat IgG1 | － |
| Goat IgG | ＋/－ |
| Rabbit IgG | ＋＋＋＋ |
| Chicken IgG | － |
Specifications
| Core | Iron Oxide (Fe3O4) |
| Layer | Dextran |
| Functional group | Protein A |
| Mean Diameter of Particles | ~1 μm |
| Storage Buffer | pH 7.4 PBS, 0.02% Tween 20 and 0.09% sodium azide. |
| Storage temp. | 2 ~ 8 ℃ |
| Capacity | 260 ug human IgG/1 ml |
Material supplied
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Beads-Protein A provides Fe3O4 beads coated with dextran of an average ~1 μm in diameter. Protein A, about 42 kDa, is coupled covalently to dextran. Beads are supplied in phosphate buffered saline, pH 7.4, containing 0.02% Tween 20 and 0.09% sodium azide.
Additional material required
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• Washing buffer: PBS buffer (pH 7.4) with 0.02% Tween 20
• Elution buffer if necessary
• Neutralization buffer if necessary
• Desired antibody
• Magntic stand: Magstand is suggested for the best performance
• Tilt rotation device or vortexer
• Eppendorf tubes & pipett
Protocol
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Protocol Preparation of beads-Protein A for use
• Magnetic stand: Magstand for the best performance
• Tilt rotation device or vortexer
• Eppendorf tubes & pipette
1. Resuspend the beads-Protein A thoroughly by pipetting or vortexing the vial for 15 secs.
2. Transfer an adequate amount of beads-Protein A into a clean tube.
NOTE: Take an appropriate amount of beads according to the binding capacity mentioned in “Binding Characteristics” above.
For example, if 2~3 μg of antibody is used in an immunoprecipitation test, 5~10 μL of beads-Protein A is enough for one test.
Exceeding the recommended amount of beads may cause high background in some cases.
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 Washing 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.
NOTE: beads-Protein A contains 0.09% NaN₃, so we strongly recommend washing the beads at least three times before use.
Binding of Antibody
10. Mix an appropriate amount of antibody (you may start with 2 μg of antibody for the preliminary test) in 200 μL Washing buffer and transfer to the tube from step 9. Then vortex for 10 secs.
11. Incubate with tilt rotation for 30 minutes at room temperature.
12. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
13. Discard the supernatant, then remove the tube from the magnetic stand.
14. Add 200 μL Washing buffer and resuspend the beads by pipetting.
15. Place the tube on the magnetic stand for 30-60 seconds to immobilize the beads at the tube wall.
16. Discard the supernatant, then remove the tube from the magnetic stand.
17. Repeat steps 14-16 two more times to remove unbound antibody.
Immunoprecipitation for Target Antigen
18. Add at least 100 μL cell lysate sample containing the target antigen to the tube from step 17. Then vortex for 10 secs.
19. Incubate with tilt rotation for 30 minutes at room temperature or at 4℃ overnight.
20. Repeat steps 14-16 three times.
21. If necessary, proceed to “SDS-PAGE analysis and Western Blot analysis” or “Elution of target antigen”.
SDS-PAGE and Western Blot Analysis
1. Mix the appropriate SDS-PAGE loading buffer with the beads from step 20, and heat to 95℃ for 5 min.
2. Directly load the mix of beads and buffer solution into the well of SDS-PAGE, then proceed with the standard SDS-PAGE and Western Blot analysis.
Elution of Antibody / Target Antigen
1. Add 20 μL elution buffer (e.g., 0.1 M Glycine-HCl, pH 2.0) after step 20, and gently mix the beads-antibody-antigen complex by pipetting.
2. Incubate with tilt rotation for 2 minutes at room temperature.
3. Place the tube on the magnetic stand for 30-60 seconds.
4. Collect the supernatant into a clean tube, then adjust the pH by adding 2 μL neutralization buffer (e.g., 1M Tris-HCl, pH 8.5).
Troubleshooting
| Troubles | Solutions |
| Immunoglobin binding is low. | 1. Ensure that the beads are thoroughly suspended before use. 2. Thoroughly and continuously mix the beads and sample using either a tilt rotation device or a vortexer. 3. Refer to Table 1 to match Protein A’s binding preferences with various immunoglobulins. 4. Optimize incubation time and temperature based on the sample volume and the antibody’s affinity for the target antigen. |
| Non-specific and background binding is high. | 1. Reduce the amount of beads used per test according to the binding capacity mentioned in "Binding Characteristics". 2. After incubating the beads-antibody complex with the antigen (Step 19), increase the number of wash steps (Step 20) to five. 3. Increase the concentration of Tween 20 to 0.1% in the washing buffer before eluting the sample. 4. Ensure that the washing buffer is completely removed. |
| Beads do not collect on the magnet. | 1. Make sure the tube is in direct contact with the magnet. 2. Use the Magstand for best performance. |