Hi, Try using 100mM Ammonium Citrate pH 8.5 as the buffer for your lysis -add salts as well. The Citrate will inhibit metalloproteases and is Ni-NTA friendly. It solved our proteolysis problems and may help with yours.
Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York 85 St. Nicholas Dr. CDI 12308 New York, NY 10031 (212) 650-6070 www.khayatlab.org<http://www.khayatlab.org> On May 19, 2015, at 2:05 AM, Manjula Ramu <manjula....@gmail.com<mailto:manjula....@gmail.com>> wrote: Thanks all for the suggestions. @ Pius, I used bacterial expression system.Yes I centrifuged precipitated protein sample and found more amount in soluble form itself, however within a day protein gets degraded. If I have to further purify the protein I have to concentrate the IMAC elutes and concentrate using filters. In this step protein degradation was more and filter used to block and could not complete the process . Do you use batch method of elution or gradient??? Also do you add PMSF in the elution buffer??? Yes Nikhil I did batch elution and included PMSF in all the buffers. and I tried using 500mM NaCl too but no change was observed. Thanks and Regards, Manjula R Research Scholar Department of Biophysics National Institute of Mental Health and Neurosciences Bengaluru-29, Karnataka INDIA E-mail: manjula....@gmail.com<mailto:manjula....@gmail.com> Mobile no:+91-9538553356 http://www.nimhans.kar.nic.in/ <https://www.nimhans.kar.nic.in/> On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN <bhanu.hydpri...@gmail.com<mailto:bhanu.hydpri...@gmail.com>> wrote: Try increasing NaCl to 500 mM and glycerol to 10%..And try reduce bME to 1mM for IMAC using Ni-NTA. If your protein requires any additional co-factors (like Mg, Ca or Zn) add them as chloride salts upto 1 to 5 mM. On Tue, May 19, 2015 at 10:19 AM, Manjula Ramu <manjula....@gmail.com<mailto:manjula....@gmail.com>> wrote: Hi all, I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 mm NaCl, 5% glycerol, 0.1mM PMSFand 5mM beta ME in the buffer and performed affinity purification. Eluted with 200mM imidazole. While elution I could see slight turbid in eluted protein (I get pure protein, single band on SDS-PAGE). However, when I centrifuged I couldn't see any pellet. After some time(a day) protein got degraded. In other method I tried cation exchange purification of lysate with MES buffer pH 6.5. Here also I saw slight precipitation and later degradation. After this again I tried with MES buffer pH 5.5 and PIPES buffer pH 6.0, here also same problem I faced. Please suggest me a method where I can get stable protein. Thanks and Regards, Manjula R Research Scholar Department of Biophysics National Institute of Mental Health and Neurosciences Bengaluru-29, Karnataka INDIA E-mail: manjula....@gmail.com<mailto:manjula....@gmail.com> Mobile no:+91-9538553356 http://www.nimhans.kar.nic.in/ <https://www.nimhans.kar.nic.in/> -- B4U
