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: [email protected] 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 <[email protected]> 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 <[email protected]> > 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: [email protected] >> Mobile no:+91-9538553356 >> http://www.nimhans.kar.nic.in/ >> <https://www.nimhans.kar.nic.in/> >> > > > > -- > B4U >
