Protein purification using iron oxide nanoparticles
Materials
Iron oxide particles
Ammonium acetate buffer
Sodium chloride
Eppendorf tube
Micropipette
Magnetic stand
Purification Protocol
1. Transfer 100 µl thoroughly resuspended iron oxide particles to a
microcentrifuge tube.
2. Place the tube on a magnet until the beads have migrated to the side of
the tube and the liquid is clear.
3. Discard the supernatant.
4. Equilibrate the beads by washing with 1 ml ammonium acetate buffer
(10mM, pH 6.7)
5. Again separate the beads from the buffer using a magnet and discard the
buffer. Repeat steps 4 and 5 for 3 times.
6. Resuspend the beads in 50µl ammonium acetate buffer
7. Add the sample (sample 1) to the beads
7. Incubate at room temperature for 30min with periodic mixing
8. Place the tube on a magnet until the beads have migrated to the side of the
tube, then remove the supernatant (sample 2).
9. Wash 3 times with 500 µl ammonium acetate buffer
10. Resuspend the beads thoroughly between each washing step.
11. Place the tube on a magnet until the beads have migrated to the side of
the tube and the liquid is clear, and then discard the supernatant.
12. Add 100 µl Elution Buffer (0.8M NaCl in ammonium acetate buffer).
13. Leave the suspension for 20 minutes at room temperature with periodic
mixing
14. Place the tube on a magnet until the beads have migrated to the side of
the tube and the liquid is clear, and then discard the supernatant.
15. Transfer the supernatant (sample 3) containing the eluted protein to a
clean tube.
16. Check the coagulation activity
Coagulation activity assay
Materials
Disposable cuvettes
Synthetic clay solution
Spectrophotometer
micropipettes
1. Take 10µl of the sample and 990µl of the substrate (synthetic clay
solution).
2. Mix well using micropipette
3. Measure the absorbance at 500nm
4. Leave the samples undisturbed for 1hr.
5. Measure the absorbance at 500nm again
Blank (calibration): 1ml of water
Samples: Crude extract (sample 1)
Unbound sample (sample 2)
Bound and eluted (sample 3)
Coagulation activity (%) = initial absorbance – final absorbance X 100
initial absorbance