• Wear protective gloves, clothing, eye, and face protection. Wash hands thoroughly after handling.
• Avoid freeze/thaw cycles and centrifugation which could damage the beads.
• Vortex samples for about 10 seconds before adding magnetic beads.
• Vortex beads for about 10 seconds and mix them well with lysates containing His-tag proteins to ensure best performance.
• Elute His-tag proteins from the beads completely.

1. Ni-IDA Si-Mag magnetic beads 10 mL (10% V/V suspension solution).
2. Binding Buffer (NOTE 1) (50 mL):20mM Sodium Phosphate, 300mM NaCl, 0~20mM (NOTE 2) Imidazole, pH7.4.
3. Wash Buffer (NOTE 1) (50 mL): 20mM Sodium Phosphate, 200mM NaCl, 0~80mM (NOTE 2) Imidazole, pH7.4.
4. Elution Buffer* (20mL): 20mM Sodium Phosphate, 150mM NaCl, 500mM Imidazole, pH7.4.

NOTE 1: 8M (final concentration) of Urea should be added to the binding buffer and/or wash buffer above for purifying protein from inclusion body;

NOTE 2: The range is for information only. It should be tested and optimized by the user.

• Si-Mag Magnet (sold separately) or other magnetic racks compatible with vials used.
• Ethanol (ACS) grade (to regenerate the beads).
• Cell lysis buffer containing inhibitor of proteases such as 1 mM of PMSF (phenylmethylsulfonyl fluoride).

Protocol

1. Sample preparation. Collect cells from E. Coli, yeast or other cell cultures. Re-suspend these cells with cell lysis buffer. Lyse cells with sonication or by French press. Centrifuge the crude lysate at 14000RPM for 15 min at 4°C. If lysis buffer contains chemicals in concentrations higher that listed in the table below use    provided binding buffer to dilute the lysates.
2. Re-suspend and Pipet out minimal 50uL of Ni-IDA magnetic beads, wash twice with water using Si-Mag magnet rack (or similar magnet racks made by other vendors).
3. Mix cell lysates with the Ni-IDA magnetic beads, incubate either at 4°C or for 20 min or at room temperature for 5 min. Insert the tube into Si-Mag magnet rack for 30 seconds.
4. Remove supernatant by holding the magnet rack upside down or by pipetting.
5. Wash the beads with 1ml of Wash Solution. Make sure the beads get completely resuspended by vortexing.
6. Repeat step 5 three times.
7. Elute proteins bound to the beads by adding 100-500 uL of elution buffer and  incubating  2-3 minutes 4°C or room temperature. Make sure the beads get completely resuspended by vortexing.
8. Immobilize beads by placing tube into a magnet rack and pipette protein solution from this tube into a new clean tube.
9. Store purified protein at -20°C for a long-term storage.

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Regeneration of Ni-IDA Magnetic beads:

1. Ni-IDA Magnetic beads can be regenerated 6-8 times. Beads should be stored in 20% ethanol solution at 4°C for a long-term storage. To avoid potential cross contamination, only use the same batch of beads for the same target His-tag protein.
2. To recharge the beads, incubate beads with the stripping buffer (50mM Tris-HCl, pH7.4, 150mM EDTA) for 10 minute at room temperature. Then wash two times with 2 mL of distilled water.
3. Wash beads with 0.5M NaOH mixed with 2M NaCl for 10 minutes at room temperature.
4. Wash beads with 2 mL of distilled water three times at room temperature.
5. Recharge the beads by incubating them with 100mM of NiSO₄ for 30 minutes at room temperature.
6. Wash beads six times with 2 mL of distilled water at room temperature.
7. Store beads in 20% ethanol solution at 4°C.