Dear Sebastian,

Thank you so much for your response.

I ligated my gene of interest in pET 21D vector system and then i transformed it into BL 21 cells for protein expression, proteins are coming in inclusion bodies. so i used probe sonicator and lysis buffer to break its membrane. In protein purification, i used binding buffer (20 mM tris Hcl, 40 mM Imidazole, 300 mM nacl, 5% glycerol), Elution buffer (20 mM tris Hcl, 1 M Imidazole, 300 mM nacl) and wash buffer (I M Imidazole).

Are you not solubilizing the inclusion bodies first like by including 8 M urea or 6 M GnHCl in all the buffers. Otherwise, you will not get the target protein, but E.coli cytosolic proteins as bands from top to bottom in the SDS PAGE.

Dear Mangal,

Thank you for your suggestion, I have went through the process of Inclusion bodies solubilization protocol where I used Isolation buffer (20 mM Tris Hcl, 2 M urea, 2% Triton X-100, pH- 8) to isolate protein from pallet, Resuspension Buffer (20 mM Tris Hcl, pH-8), and Binding Buffer (20 mM Tris Hcl, 8M urea, 300 mM Nacl, 1mM BME) to isolate crude protein and then performed rest of the work.

Hi Sebastian,

Thank you for sharing your experience and protocol. your suggestion looks useful to me and I will defiantly try all the possible ways explained by you.

Your answer gave me a new way to design my protocol from the beginning.

Thanks once again.

If your protein is membrane protein, please use Brij-58 (palmitoylated non-ionic surfactant) instead of Triton-X100 (octyl-phenylated non-ionic sufactant) at 1% (v/v).

Brij-58 is working in membrane protein separation (please see file; SEC column 300 A silica). I have previously found that human bile can only be separeted on HPLC-SEC column with Brij-58, whereas bile can not be analysed by using 1% (v/v) NP-40 (octyl-phenylated non-ionic surfactant) due to gradual increase in the column-inlet pressure.

Eluent may become viscous, and please use HPLC instead of FPLC.

Dear Dr. Kou,

Thank you for your suggestion, I will try to follow the suggestion given by you.