The information about of the cleavage site can be used in two ways : possibly identify the protease - as it has already been suggested - or introduce a mutation at the cleavage site which would prevent clipping from occurring.
The caveat of introducing a mutation is that there is always the possibility that the mutated residue plays a key role. Good luck Thierry From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe A. Rice Sent: Tuesday, May 19, 2015 12:23 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein precipitation For one of our more easily-degraded proteins, we added a mix of protease inhibitors (those expensive tablets you can buy) and also put a drop of EDTA into each tube in the fraction collector so that any contaminating metal-dependent proteases would be knocked out as soon as the protein came off the metal affinity column. That seemed to help. Also, if you can get your protein to stick to the next column in whatever buffer it comes off the IMAC column in, there's no need to dialyze or concentration between columns - the faster you get it clean, the better. Good luck! Phoebe Rice ________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Michel, Max [m.mic...@fz-juelich.de] Sent: Tuesday, May 19, 2015 1:32 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Protein precipitation Hi Manjula, I had a similar problem: pH, salt, additives and protease inhibitors (I tried them all) didn't work. The protein degraded after lysis of bacteria within 20 min at 24°C to 50 %. I had luck after i cooled down all buffers and materials to less than 4 °C or used them icecold ... (1L bottle buffer stirred for 3-4h in icewaterbath). I performed IMAC, IEX and SEC to get rid of the degradation. Finally the Protein was stable for at least one month. You have to take care about temperature induced pH changes. Some buffers change their pH up to 0.5 (like Tris) when stirring from 24 to 2 °C. This can lead to precipitation if you are working close to the pI of your protein. Maybe you can try to characterize the protease to find a suitable protease inhibitor. Make some massspectrometry with your degraded sample. An other option is to test via westernblot. If you have some C- oder N-terminal monoclonal well characterized antibody. Greets Max ________________________________ Von: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK]" im Auftrag von "Manjula Ramu [manjula....@gmail.com] Gesendet: Dienstag, 19. Mai 2015 08:05 An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Betreff: Re: [ccp4bb] Protein precipitation 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 ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ Forschungszentrum Juelich GmbH 52425 Juelich Sitz der Gesellschaft: Juelich Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498 Vorsitzender des Aufsichtsrats: MinDir Dr. Karl Eugen Huthmacher Geschaeftsfuehrung: Prof. Dr.-Ing. Wolfgang Marquardt (Vorsitzender), Karsten Beneke (stellv. Vorsitzender), Prof. Dr.-Ing. Harald Bolt, Prof. Dr. Sebastian M. 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