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a third way to use the information would be to reclone the part before and/or after the protease cleavage site, i.e. you may have identified (a) stable domain(s) that may crystallize much better than the full-length protein. Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 19 May 2015, at 18:48, Fischmann, Thierry wrote: > 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:[log in to unmask]] On Behalf Of Phoebe A. Rice > Sent: Tuesday, May 19, 2015 12:23 PM > To: [log in to unmask] > 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 [[log in to unmask]] on behalf of Michel, Max [[log in to unmask]] > Sent: Tuesday, May 19, 2015 1:32 AM > To: [log in to unmask] > 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 [[log in to unmask]]" im Auftrag von "Manjula Ramu [[log in to unmask]] > Gesendet: Dienstag, 19. Mai 2015 08:05 > An: [log in to unmask] > 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: [log in to unmask] > Mobile no:+91-9538553356 > http://www.nimhans.kar.nic.in/ > > On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN <[log in to unmask]> 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 <[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] > Mobile no:+91-9538553356 > http://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. 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