Hello everyone,
Can anyone tell me about the standard protocol for On column refolding of denatured proteins (by 8M urea) using Ni IDA column? what exact concentrations of BME should be used during the procedure and incubation time n all.
Does your protein contains cysteines? If yes, add beta-mercaptoethanol at a final concentration of 1mM to all the buffers.
Washing buffer: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 6 M urea, 1 mM 2-mercaptoethanol pH 8.0
Refolding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0
Elution Buffer: 20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0
The pH of buffers are dependent upon your protein's pI. You can change the salt concentration also. Check if this works else optimize as per your requirements.Good luck.
The change from 6 M urea to 0 M urea has to be run slowly. If you know what you are doing you will realize that the pH of buffers is independent of the pI of the protein. Do not reduce salt concentration below 500 mM.
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