1. Overview

His-Tag protein purification magnetic beads (Magarose-IDA/NTA/TED) is a new purification medium developed for the purification of His-tagged proteins, which has the characteristics of high efficiency, speed and convenience, and can directly purify high-purity target proteins from biological samples in one step through magnetic separation, greatly simplifying the purification process and improving purification efficiency, and is suitable for convenient purification of His-tag proteins in scientific research and industry. Compared with the traditional column chromatography purification method, Magarose NTA magnetic beads purify histidine-tagged proteins through magnetic separation, eliminating the need for multiple time-consuming and laborious centrifugation and filtration steps for crude protein samples, no need for column loading and column chromatography, and no need for expensive column chromatography equipment.

This product range includes two metal ion chelated magnetic beads, nickel and cobalt. They differ in the amount of target protein binding and the purity of the target protein, and it is generally recommended that customers use nickel ion chelated magnetic beads, which can meet the customer's requirements for protein load and purity in most cases. Cobalt ion chelated magnetic beads are recommended for customers with higher purity requirements.

His-Tag Protein Purification Magnetic Beads (Magarose-IDA/NTA/TED) Magnetic beads are widely used for the purification of soluble histidine-tagged proteins secreted or intracellularly expressed in bacterial, yeast, insect, and mammalian cells, as well as denatured proteins.

2. Product characteristics

Product Name	Magarose-IDA/NTA/TED
Item number	PMAG001-1/001-2/001-3
Magnetic bead concentration	25%(v/v)in 20% ethanol
ligand and density	IDA or NTA or TED
Medium	6% cross-linked magnetic agarose
Particle size	20-45μm
Ligand binding	30-40mg fusion protein/ml magnetic beads
Save	Store at 2~8°C for one year

Note: IDA has high binding capacity and poor chelating agent resistance. NTA binding capacity is high, and chelating agent resistance is better. TED has low binding capacity and excellent resistance to chelating agents.

3. How to use

1. Recommended buffer

A. Buffers and formulations required for soluble histidine-tagged protein purification

Binding Buffer: 20mM PBS, 500mM NaCl, 5~20mM imidazole, pH7.4;

Wash Buffer: 20mM PBS, 500mM NaCl, 20~100mM imidazole, pH7.4;

Elution Buffer: 20 mM PBS, 500 mM NaCl, 300-500 mM imidazole, pH 7.4;

B. Buffers and formulations required for inclusion histidine-tagged protein purification

Binding Buffer: 8M Urea, 50mM NaH₂PO₄, 100mM Tris· HCl, pH8.0;

Wash Buffer: 8M Urea, 50 mM NaH₂PO₄, 100 mM Tris· HCl, pH6.3;

Elution Buffer: 8 M Urea, 50 mM NaH₂PO₄, 100mM Tris· HCl, 300-500 mM imidazole, pH4.5;

Note: Buffers need to be filtered and sterilized with a 0.22 or 0.45 μm membrane before use. The recommended buffer system is suitable for the purification of most histidine-tagged proteins, and adding a certain concentration of imidazole to the binding buffer can reduce non-specific binding and improve the purity of the protein of interest. First-time customers can use the recommended buffer and adjust the concentration of imidazole in the buffer appropriately according to the experimental results.

2. Sample preparation

2.1 Histidine-tagged protein sample preparation (soluble expression under native conditions under E. coli).

Dilute the recombinant expressed proteins: add 5~10 ml binding buffer per gram of E. coli bacteria and resuspend it. The sample and binding buffer containing the same concentration of imidazole avoided the histidine-containing protein expressed by the host cell. At the same time, large particles and high concentrations of chelating agents, such as EDTA, histidine, citric acid and other impurities, should be removed to prevent the destruction of Ni-NTA resin.

1. Enzymatic hydrolysis: 0.2 mg/ml lysozyme, 20 μg/ml DNase, 1 mM MgCl2, 1 mM PMSF (final concentration) or other protease inhibitors. The added inhibitor must have no effect on the magnetic beads and be mixed at 4 °C for 30 min.

2. Mechanical cracking: ultrasound, homogenization, repeated freeze-thaw, etc.

3. Adjust the pH of the lysate to 7.4: Do not adjust the pH with strong bases or acids (to prevent precipitation).

4. Centrifugation of the lysate: Transfer the liquid to a centrifuge tube for 20 min at room temperature or 4°C at 12000 rpm, the choice of temperature depends on protein stability.

5. Collect the supernatant for subsequent experiments, or collect the precipitate after centrifugation to prepare for the next experiment.

6. For proteins expressed in yeast, insect and mammalian cell systems, if there is a large amount of supernatant, ammonium sulfate can be used for protein precipitation. Dialysis with 1X PBS, add to the magnetic beads, if the sample does not contain EDTA, histidine, citric acid and other components that affect the magnetic beads, it can be directly placed on the column.

2.2 Histidine-tagged protein sample preparation (purification under denaturing conditions for inclusion expression in E. coli).

1. Suspend cells with 1× PBS (approximately 5 ml of 1× PBS per ml of bacterial pellet) and perform the experiment according to the ultrasonic conditions above.

2. Centrifuge the lysate at 12,000 rpm for 10 min to collect the inclusions. Wash the inclusions several times with 1× PBS.

3. Dissolve the inclusions with a binding/wash buffer (about 5 ml of inclusions per ml) and incubate at room temperature for 30~60 min. Homogenization or ultrasound may be required to completely dissolve the pellet.

4. Centrifuge at 12,000 rpm for 30 min to remove any remaining insolubles. Carefully transfer the supernatant to a clean tube without touching the pellet underneath.

2.3 Histidine-tagged protein purification steps

The amount of magnetic beads used is calculated by the user based on the target protein yield and magnetic bead load information, here is an example of obtaining 5-10mg of target protein:

1. Magnetic bead preparation: Mix the Ni-NTA magnetic beads thoroughly, use a pipette to take 2 ml of magnetic bead suspension, (magnetic bead sedimentation volume 500μl) in a centrifuge tube, magnetically separate, wait for the solution to be clarified, aspirate the clear solution, add_ddH2Oand wash repeatedly to remove alcohol.

2. Magnetic bead balance: Add 5 ml Binding Buffer, blow repeatedly 5-10 times, magnetically separate, wait for the solution to be clarified, absorb the clear solution, and wash twice.

3. Magnetic beads bind to the target protein: use 10 ml Binding Buffer to suspend 2 g of wet heavy bacteria, break and lyse it, that is, the target protein coarse sample, add the crude protein crushing solution to the magnetic beads, and mix upside down. Incubate at room temperature for 15 min (if the target protein is unstable, incubate at 2-8°C for 30 min).

4. Washing impurities: Place the centrifuge tube in the magnetic separator, wait for the solution to clarify, remove the supernatant, and keep it for sampling and testing. Add 10 ml Wash Buffer to the centrifuge tube, blow repeatedly 5-10 times, magnetically separate, wait for the solution to clarify, aspirate the supernatant, and retain for testing. Repeat the impurity washing step more than 3 times.

5. Elution: Users can change the elution volume to adjust the target protein concentration as needed, add 2-10 ml Elution Buffer to the centrifuge tube, resuspend the magnetic beads and mix well, magnetically separate, wait for the solution to clarify, and aspirate the supernatant, which is the target protein component. The above steps were repeated several times to collect the eluting components and leave samples for testing to increase the recovery of the protein of interest.

6. Post-treatment of magnetic beads: Add 5 ml of Elution Buffer, resuspend the magnetic beads, separate the magnets, aspirate the supernatant, and repeat the step twice. Wash repeatedly with 5 ml_ddH2O3-5 times. Finally, add 2 ml of 20% ethanol solution so that the total volume is equal to the volume of the initial magnetic bead suspension and store at 2-8 °C.

7. SDS-PAGE detection: The purified samples are detected with SDS-PAGE.

8. Magnetic bead regeneration: If magnetic beads are used more than three times in a row, their ability to bind to the target protein may be significantly reduced, and magnetic bead regeneration is recommended.

Stripping Buffer: 20 mM Sodium Phosphate, 500 mM NaCl, 100 mM EDTA, pH 7.4

Washing Buffer (optional): 0.5 M NaOH, 2 M NaCl

Recharge Buffer: 100 mM NiSO₄ (this chemical agent is toxic and may cause allergic reactions, so be careful when using it) Take 5 mL of 10% (v/v) magnetic bead suspension as an example to detail the magnetic bead regeneration operation:

1) Magnetically separate the magnetic bead suspension, remove the supernatant, add 5 mL ddH2O to the centrifuge tube, resuspend the magnetic beads, and magnetically separate to remove the supernatant.

2) Add 5 mL of Stripping Buffer, resuspend the magnetic beads, mix at room temperature for 5 min, magnetically separate, and remove the supernatant. Repeat this step 1 time.

3) Add 5 mL of_ddH2O, resuspend the magnetic beads, magnetically separate to remove the supernatant, and repeat this step twice.

4) Alkali treatment: Add 5 mL of Washing Buffer, resuspend the magnetic beads, rotate and mix at room temperature for 5 min, magnetically separate, and remove the supernatant. Add 5 mL of_ddH2O, resuspend the beads, magnetically separate, and remove the supernatant. Repeat the_ddH2Owashing step 3~5 times until the washing solution is neutral.

5) Add 5 mL of Recharge Buffer, resuspend the magnetic beads, rotate and mix at room temperature for 20 min, remove the supernatant, and remove the supernatant.

6) Add 5 mL of ddH2O, resuspend the beads, magnetically separate and remove the supernatant. Repeat this step more than 4 times to ensure complete nickel removal.

7) Add 2ml of 20% ethanol solution so that the total volume is equal to the volume of the initial magnetic bead suspension, and store at 2-8°C.

3. Precautions

1) Freezing, drying, and high-speed centrifugation should be avoided during the use and storage of magnetic beads.

2) Before using the product, be sure to fully oscillate the beads to make the beads suspended evenly;

3) During the mixing process of magnetic beads and solution, if the solution is viscous and cannot be resuspended by flipping the centrifuge tube, the magnetic beads can be fully resuspended by repeated blowing with a pipette or by swirling for a short time;

4) Users can retain the supernatant removed by magnetic separation for sampling and testing according to actual needs, so as to analyze the purification process and optimize the protein purification process;

5) It is recommended to purify the same protein when this product is used repeatedly, and when purifying different proteins, it is recommended to use new magnetic beads to prevent cross-contamination;

6) This product needs to be used with a magnetic separator;

7) This kit is only used for in vitro experiments, and cannot be used for clinical, therapeutic, and in animal experiments, and is not responsible for the consequences arising therefrom.

More protein purified magnetic beads: http://www.purimagbead.com/Product/Proteinextract/

Customer paper references:

[1] Zhang Haozhe. Serum-assisted PD-L1 aptamer screening and robust characterization[D].Beijing University of Chemical Technology,2022.DOI:10.26939/d.cnki.gbhgu.2022.001916. (Ni magnetic bead purification protein)
[2] Peng Xueyu. Hunan University,2022.DOI:10.27135/d.cnki.ghudu.2022.000204. (Ni magnetic bead purification protein)
[3] Liu Fuhao, Fan Yangen, Wang Yu, et al. Screening and identification of CsHIPP26.1 protein chelating ions in tea plant golden buds[J].Tea Science,2022,42(02):179-186.DOI:10.13305/j.cnki.jts.2022.02.006. (Ni magnetic bead purified protein)

[4] Peng X, et al. DNA Nanostructure-Programmed Cell Entry via Corner Angle-Mediated Molecular Interaction with Membrane Receptors. Nano Lett. 2021 Aug 25; 21(16):6946-6951. doi: 10.1021/acs.nanolett.1c02191. Epub 2021 Aug 16. PMID: 34396773. (Si-IDA-Ni magnetic bead purified protein)

[5] Wang, J., et al. Characterization of efficient xylanases from industrial-scale pulp and paper wastewater treatment microbiota. AMB Expr 11, 19 (2021). https://doi.org/10.1186/s13568-020-01178-1 (Ni magnetic bead purified protein)

[6] Ma X, et al. Switch-on Fluorescence Analysis of Protease Activity with the Assistance of a Nickel Ion-Nitrilotriacetic Acid-Conjugated Magnetic Nanoparticle. Molecules 2023, 28, 3426. https://doi.org/10.3390/molecules28083426 (NTA-Ni binding to His6-terminal polypeptide)

[7] Liu Yiyang, Chen Huating, Liu Chunfeng, et al. Heterologous Expression, Enzymatic Properties Analysis of Key Protease GME238_g of Aspergillus oryzae BL18 and Its Application in Soy Sauce[J/OL].Food and Fermentation Industry,1-12[2025-04-23].https://doi.org/10.13995/j.cnki.11-1802/ts.042000.