Editing T7 High Yield In vitro Translation (section)

== Purification of His- or HQ-Tagged Recombinant Protein Using Magnetic Ni-Particles ==

Materials

• magnetic stand (see Section 10.C for options)

• Magnetic Ni Beads

• solid NaCl or 5M NaCl solution

1. Add 50µl of Binding/Wash Buffer to a 50µl reaction containing the His-tagged protein. [For HQ-tagged protein, also add NaCl (500mM final concentration) to improve binding of HQ tag to magnetic Ni-Particles.]

2. Vortex the magnetic Ni-Particles to obtain a uniform suspension.

3. Add 30µl of magnetic Ni-Particles.

Note: You may need to titrate the amount of magnetic Ni-Particles used for proteins expressed at higher or lower levels.

4. Invert tube to mix (approximately 10 times), and incubate for 2 minutes at room temperature. Make sure the magnetic Ni-Particles are well mixed.

5. Place the tube in the appropriate magnetic stand for approximately 30 seconds to capture the magnetic Ni-Particles. Using a pipette, carefully remove the supernatant.

6. Remove the tube from the magnetic stand. Add 150µl of&nbsp;magnetic Binding/Wash Buffer with 500mM NaCl to the magnetic Ni-Particles, and pipet to mix. Make sure that particles are resuspended well.

7. Place the tube in the appropriate magnetic stand for approximately 30 seconds to capture the magnetic Ni-Particles. Using a pipette, carefully remove the supernatant.

8. Repeat the wash step twice for a total of three washes.

9. Remove the tube from the magnetic stand. Add 100µl of magnetic Elution Buffer, and pipet to mix. Note: HQ-tagged proteins may elute at a lower concentration of imidazole (50–100mM) compared to polyhistidine-tagged proteins. The magnetic Elution Buffer, which contains 500mM imidazole, can be diluted with magnetic Binding/Wash Buffer or water to decrease the imidazole concentration, if desired.

10. Incubate for 1–2 minutes at room temperature. Place tube in a magnetic stand to capture the&nbsp;magnetic Ni-Particles. Using a pipette, remove the supernatant containing the purified protein.

Analyze the samples by SDS-PAGE or by functional assay. Note: Steps 8–10 can be omitted, and the sample containing the magnetic Ni-Particles can be used directly for SDS-PAGE analysis. Add 30µl of 1X SDS gel-loading buffer to resuspend the magnetic Ni-Particles. Load the sample directly onto an SDS-polyacrylamide gel.
[[Category:Protocols]]