Hi Manjula,
I will lyse the bacterials cells and monitor the degradation of the lysed protein in lysis buffer over 4-5 days before proceeding with purification. What is the lifetime of this expressed protein once lysed? How long can it stay after lysis without degradation or aggregation?
Does this protein contain disulfide bonds? How many? How big is this protein?
Smita Smita Mohanty, Ph.D. Associate Professor Department of Chemistry447 Physical SciencesOklahoma State UniversityStillwater, OK 74078-3071 Phone: (405) 744 6636E-mail: [log in to unmask]: chemistry.okstate.edu/mohanty
On Monday, May 18, 2015 11:49 PM, Manjula Ramu <[log in to unmask]> 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: [log in to unmask] no:+91-9538553356
http://www.nimhans.kar.nic.in/
