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Starose Mag Ni-NTA/TED—A Tool for Rapid High-throughput Screening of His-tagged Proteins

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  • Time of issue:2022-07-20 15:46
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(Summary description)

Starose Mag Ni-NTA/TED—A Tool for Rapid High-throughput Screening of His-tagged Proteins

(Summary description)

  • Categories:News
  • Author:
  • Origin:
  • Time of issue:2022-07-20 15:46
  • Views:
Information

Starose Mag Ni-NTA/TED

 

Starose Mag Ni-NTA and Starose Mag Ni-TED are agarose magnetic bead products for protein purification. They are superparamagnetic and can achieve high-efficiency targeting of histidine (His)-tagged proteins under the action of an external magnetic field. Purification and screening.

Starose Mag Ni-NTA/TED enables simple and rapid purification and screening of His-tagged proteins. After the sample is fully incubated with the magnetic beads, the magnetic beads are quickly captured by a magnetic device, the supernatant is discarded, impurities such as unbound proteins are separated from the target protein, and the His-tagged protein is removed from the magnetic beads by appropriate elution conditions. Elution completes the purification. The processing time of a single sample can be completed in as little as 15 minutes.

 

Figure 1. Process for the purification of His-tag Pro using Starose Mag Ni-NTA/TED

Table 1. Starose Mag Ni-NTA/TED performance parameters

 

Starose Mag Ni-NTA Performance Verification

 

Feature 1: Efficient enrichment and purification of low-concentration samples

 

Figure 2. Sample purification at different concentrations

 

Summary: Dilute the same total amount of His-tagged protein samples to different volumes with equilibration buffer and purify them separately using Starose Mag Ni-NTA. It can be seen from the results that even when the sample content is 0.05 mg/mL, 100 μL of magnetic beads can still maintain a yield of > 70%, and after the final purification of different samples, the purity of the target protein is always maintained at more than 97%.

 

The results show that Starose Mag Ni-NTA magnetic beads can still maintain high recovery and purity for low abundance samples.

 

Feature 2: Excellent binding efficiency

Figure 3. Elution after different incubation times

 

Summary: Take Starose Mag Ni-NTA magnetic beads, incubate them with the same volume and concentration of His-tagged protein, and take samples every 5 minutes for elution and purification. It can be seen from the results that the protein yield can reach 70% when the incubation time is only 5 min. With the increase in the incubation time, the yield is significantly improved. When the incubation time reaches 15 min, the yield increases to 90%. As the incubation time continued to increase, the yield did not increase significantly. And after incubation at different times, the purity of purified protein samples was not significantly different (both higher than 98%).

 

The results show that: Starose Mag Ni-NTA magnetic beads can quickly capture the target protein, the magnetic beads have a strong capture ability, and are suitable for the capture of unstable proteins and application scenarios that require rapid operation; and with the increase of sample incubation time, there is no Additional non-specific adsorption ensures high purity in pursuit of high recovery.

 

Feature 3: The purity of the eluted protein can be optimized and adjusted through the purification process

Figure 4. Imidazole equilibration, incubation and purification of different concentrations

 

Summary: The purification of His-tagged proteins was performed after equilibration of Starose Mag Ni-NTA with equilibration buffers of different imidazole concentrations. It can be seen from the results that as the concentration of imidazole in the equilibrium solution increases, although the yield decreases significantly, the purity gradually increases.

 

The results show that the purification effect of Starose Mag Ni-NTA magnetic beads can be adjusted to meet the final requirements, but the trade-off between purity and yield must be balanced.

 

Feature 4: Stable process repeatability

Figure 5. 6 replicates of the Starose Mag Ni-NTA purification process

 

Summary: Six process replicates were performed on the same His-tagged protein using Starose Mag Ni-NTA. It can be seen from the results that the same process was used for 6 repeated purifications, and the purity of each eluted sample was maintained at more than 98%, and the yield was maintained at 90%-93% (using the same magnetic beads for the same protein sample) To improve the stability of the experimental results during purification, it is necessary to ensure that the magnetic beads have sufficient time to interact with the magnet in each purification process, and at the same time, be careful not to absorb the magnetic beads when sucking the liquid, which will reduce the number of magnetic beads and affect the yield), relatively stable.

 

The results show that Starose Mag Ni-NTA has a stable effect of repeated experiments without cleaning and regeneration.

 

Feature 5: Process amplification is stable

 

Figure 6. Scale-up of the purification process

 

Summary: 10x scale-up of the same His-tagged protein purification process using Starose Mag Ni-NTA. It can be seen from the results that the purities of the elution samples were 98.2% and 98.5%, and the yields were 90.5% and 89.1%, respectively, before and after the process was scaled up.

 

The results show that Starose Mag Ni-NTA can realize the amplification of the purification process of the same protein sample without affecting the purity and yield.

 

Starose Mag Ni-TED Performance Verification

 

Feature 1: Good tolerance to EDTA

 

Figure 7. EDTA tolerance at different concentrations

 

Summary: Usually secreted and expressed recombinant proteins have a low concentration in the supernatant. To avoid degradation of the target protein, EDTA is often added as a metalloproteinase inhibitor. EDTA can detach the metal ion ligands coupled with the conventional Ni-NTA magnetic beads/medium, so the conventional His-tag affinity magnetic beads/medium is very sensitive to EDTA. Starose Mag Ni-TED is highly resistant to EDTA. 0, 2, and 10 mM EDTA were added to the Sf9 insect cell culture medium that secretes and expresses His-tagged protein, and Starose Mag Ni-TED was used to purify. the difference.

 

The results show that Starose Mag Ni-TED works well in media containing at least 10 mM EDTA, a concentration higher than that found in most enzyme inhibitor cocktails.

 

Feature 2: Good tolerance to EDTA

 

Figure 8. DTT tolerance at different concentrations

 

Summary: DTT is reductive and can be used to prevent intramolecular or intermolecular disulfide bonds formed between cysteines in proteins. DTT can reduce the metal ions on the conventional Ni-NTA magnetic beads/media to brown, which affects the effect of binding to proteins. Starose Mag Ni-TED is highly resistant to DTT. Add 0, 1, and 5 mM DTT to the medium of Sf9 insect cells that secrete and express His-tagged protein, respectively, and use Starose Mag Ni-TED for purification. obvious difference.

 

The results show that: Starose Mag Ni-TED can be used normally in a medium containing at least 5 mM EDTA (generally, 1-2 mM DTT can play a role in protein protection).

 

Feature 3: The purity of the eluted protein can be optimized and adjusted through the purification process

 

Figure 9. Imidazole equilibration, incubation, and purification at different concentrations

 

Summary: The purification of His-tagged proteins was performed after equilibration of Starose Mag Ni-TED with equilibration buffers with different imidazole concentrations. It can be seen from the results that as the concentration of imidazole in the equilibrium solution increases, although the yield decreases significantly, the purity gradually increases.

 

The results show that the purification effect of Starose Mag Ni-TED magnetic beads can be finally achieved by adjusting the process to balance the requirements of purity and yield.

 

Feature 4: Stable process repeatability

Figure 10. 6 replicates of the Starose Mag Ni-TED purification process

 

Summary: Six process replicates were performed on the same His-tagged protein using Starose Mag Ni-TED. It can be seen from the results that the same process was used for 6 repeated purifications, and the purity of each eluted sample was maintained at more than 97%, and the yield was maintained at 89% to 92% (using the same magnetic beads for the same protein samples. To improve the stability of the experimental results during purification, it is necessary to ensure that the magnetic beads have sufficient time to interact with the magnet during each purification process, and at the same time, be careful not to absorb the magnetic beads when sucking the liquid, which will reduce the number of magnetic beads and affect the yield), relatively stable.

 

The results show that Starose Mag Ni-TED has a stable effect of repeated experiments without cleaning and regeneration.

 

Feature 5: Process scale-up is stable

Figure 11. Scale-up of the purification process

 

Summary: The purification process of the same His-tagged protein was amplified by 10 times using Starose Mag Ni-TED. It can be seen from the results that the purities of the eluted samples were 99.1% and 99.3%, and the yields were 90.0% and 89.8%, respectively, before and after the process was scaled up.

 

The results show that Starose Mag Ni-TED can realize the amplification of the purification process of the same protein sample without affecting the purity and yield.

 

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