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Ni-NTA magnetic beads purification His-tag protein purification using Ni-NTA magnetic beads


Materials and reagents

Extraction buffer

20 mM Tris-HCl, pH 8.0

1 mM PMSF

1 mg/ml lysozyme

0.05% Triton X-100



Magnetic PBS

10 mM potassium phosphate, pH 7.2

250 mM NaCl

0.05% Triton X-100



Washing buffer

10 mM KP, pH 7.8

300 mM NaCl

20 mM imidazole

8% glycerol

0.2% Triton X-100



Materials

Ni-NTA magnetic beads (Qiagen)
1.5-ml microfuge tubes
sample mixer equiped with tube-holding wheel (Dynal)
magnetic rack (Dynal MPC-S)
Ultrasonic water bath (Branson 200)



Preparation of the magnetic beads

1. Pipette 30 ml of the magnetic suspension from the bottle and apply it to a 1.5-ml microfuge tube.
2. Place the tube in the magnetic rack, apply the magnet and remove the supernatant.
3. Wash the beads with 300 ml magnetic PBS.
4. Resuspend the beads in 50 ml extraction buffer.



Procedure


1. Apply 1.3 ml of bacterial culture to a microfuge tube and spin for 5 min at 13,000 rpm. Store the pellet at –20°C
2. Add 300 ml of extraction buffer to the pellet, resuspend the cells and incubate the suspension for 30 min at room temperature.
3. Homogenize by sonicating the cell suspension for 3 min in the ultrasonic water bath.
4. Take 10 ml of the homogenate and store for SDS-PAGE analysis. Centrifuge the rest for 10 min at 13,000 rpm.
5. Remove the supernatant and discard the pellet. Take a 10-ml sample of the supernatant for SDS-PAGE analysis.
6. Pipette the rest of the supernatant to the prepared magnetic beads (in a 1.5-ml microfuge tube) and incubate the tube in the rocking device for 30 min at room temperature.
7. Apply the magnet and remove the supernatant.
8. Wash the beads with 300 ml washing buffer for 30 min at room temperature using the rocking device.
9. Then, wash the beads with 300 ml of PBS for 10 min.
10. Pipette 12 ml SDS sample buffer to the beads and boil for 3 min.
11. Mix the homogenate and supernatant samples (10 ml) with 10 ml SDS sample buffer and boil for 3 min.
12. Analyze the samples [homogenate (total protein), supernatant (soluble protein) and purified protein] by SDS-PAGE.


Last updated: 8 May 2001