SweMagrose IDA-Ni Magnetic Agarose Beads for His-Tag Protein Purification

Product Information

Product Description/Introduction

This product is prepared from agarose magnetic beads modified by IDA and chelating nickel ions. It can efficiently and quickly purify the histidine-tagged proteins in biological samples without centrifugal filtration. The operation is simple and suitable for the purification of soluble histidine tag proteins expressed in the supernatant or cells of bacteria, yeast and cells. it can also be combined with high-throughput protein purification equipment for protein screening and separation.

Ni2+ has 6 ligands and strong protein binding ability, Co2+ has 4 ligands and less non-specific adsorption, for higher purity requirements, you can choose our other SweMagrose IDA-Co Magnetic Agarose Beads for His-Tag Protein Purification (G3655).

Storage and Shipping Conditions

Ship with wet ice; Store at 4，valid for 12 months.

Product Components

Assay Protocol/Procedures

1. The configuration of various reagents can be referred to as follows. Users can choose the appropriate amount of magnetic beads and buffer formula according to the situation of the reagents.

2. Buffer configuration

a) The binding performance of the target protein with metal ion chelating beads will directly affect the purification efficiency of the target protein, and various buffers will also affect the recovery and purity of the target protein to some extent. Therefore, before large-scale protein purification, users should design their own experiments to screen the buffers suitable for the target protein, including binding buffer, washing buffer and elution buffer.

b) The buffer system provided below is suitable for the purification of most histidine tag proteins for users' reference.

Binding Buffer：20 mM Sodium Phosphate，500 mM NaCl，5~50 mM Imidazole，pH7.4

Wash Buffer：20 mM Sodium Phosphate，500 mM NaCl，50~100 mM Imidazole，pH7.4

Elution Buffer：20 mM Sodium Phosphate，500 mM NaCl，500 mM Imidazole，pH7.4

3. Sample treatment

a) E. coli, yeast and other intracellularly expressed proteins: add 5~10 mL of Binding Buffer per gram of cells, add protease inhibitor (e.g., PMSF at a final concentration of 1 mM), resuspend the cells, and ultrasonically lysed the cells in an ice bath, i.e., the crude protein sample. If the sample is too viscous, appropriate amount of nuclease can be added to the crude sample as needed and placed on ice for 30 min to degrade the nucleic acids. Alternatively, centrifugation of the protein sample can be performed as needed.

b) Extracellular expressed protein: The crude protein sample was obtained when the extracellular expression supernatant was diluted with an equal amount of Binding Buffer.

c) Intracellularly expressed proteins in animal cells: Take appropriate amount of animal cells, wash with appropriate amount of PBS (self-provided) once, discard the supernatant, resuspend with appropriate amount of Binding Buffer containing 1% (v/v) Triton X-100 or 1% (v/v) NP-40, add protease inhibitor (e.g., PMSF at a final concentration of 1 mM), and put it on ice for 10 min, that is crude protein sample.

4. Binding of target protein to magnetic beads:

a) Suspend 2 g wet weight of the organisms with 10 mL of Binding Buffer, and after fragmentation and lysis, a sample of the target crude protein is added to a centrifuge tube containing pre-treated magnetic beads, and the tube is placed in a vortex mixer and shaken for 15 s. The target crude protein sample is then added to a centrifuge tube with pre-treated magnetic beads.

b) Place the centrifuge tube on a rotary mixer for 20-30 min at room temperature (if needed, can rotate and mix for 1 h at a low temperature of 2-8°C to prevent degradation of the target protein).

c) Place the centrifuge tube on the magnetic separator for separation, transfer the supernatant to a new centrifuge tube for subsequent testing, and remove the centrifuge tube from the magnetic separator for subsequent washing steps.

5. Magnetic bead washing:

a) Add 10 mL of Wash Buffer to the centrifuge tube containing the magnetic beads, gently turn the tube several times to resuspend the beads, separate them magnetically, and transfer the wash solution to a new centrifuge tube for sampling. Repeat this procedure once.

b) Add 10 mL of Wash Buffer to the centrifuge tube containing magnetic beads to resuspend the beads, transfer the bead suspension to a new centrifuge tube (to avoid contamination of target proteins by non-specifically adsorbed proteins on the wall of the original centrifuge tube), magnetically separate, and pipette the supernatant into the Wash Buffer Collection Tube.

6. Target protein elution

a) Users can adjust the concentration of target protein by changing the elution volume as needed. Add 2-10 mL of Elution Buffer, gently turn the tube several times to suspend the beads, magnetically separate the beads, and collect the eluate into a new centrifuge tube, which is the purified target protein sample.

a) If necessary, repeat the above steps once to collect the sample into a new centrifuge tube to test whether the target protein is eluted completely.

b) Detect the target protein by SDS-PAGE or Western blotting. If you need to measure the protein concentration, you can use Elution Buffer to zero the concentration, or use dialysis or ultrafiltration to remove Imidazole and then measure the concentration.

7. Magnetic bead post-processing

a) Add 5 mL of Elution Buffer to the centrifuge tube containing the magnetic beads, turn the tube up and down several times to suspend the beads, separate them magnetically and remove the supernatant.

b) Repeat the above steps twice.

c) Add 5 mL of ddH2O to the centrifuge tube containing the magnetic beads, turn the tube up and down several times to suspend the beads, separate them magnetically and remove the supernatant.

d) Repeat the above steps twice.

e) Add the preservation solution to the magnetic beads to make a total volume of 5 mL, store at 2-30°C (for long-term storage, place at 2-8°C), and can be used for the next purification of the same protein.

8. Magnetic bead regeneration

When the magnetic beads are used continuously for 3 or more times, the ability to bind target proteins may be significantly reduced, and it is recommended to carry out the magnetic bead regeneration process. Take 5 mL of 10% (v/v) magnetic bead suspension as an example to illustrate the magnetic bead regeneration operation in detail.

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