In continuation of this thread, what is the success rate of separating TEV and MBP tag from my desired protein after TEV cleavage using Hydrophobic/Ion exchange column? I have also read TEV is less active in a high concentration of Imidazole, so dialysis before adding TEV may work better but then reaction buffer will have 0.5mM EDTA and 1mM DTT, can these affect Hydrophobic/Ion Exchange resin? (EDTA and DTT certainly not good for Affinity resins though)
Regards Nishant On Sat, Nov 18, 2017 at 2:19 AM, Anshumali Mittal <anshumali...@gmail.com> wrote: > Hi Liuqing, > > The scheme you suggested (or heard) of using Ion-exchange at the end can > be useful to separate protein with same net charge but different > conformations. > > Ciao > > On Fri, Nov 17, 2017 at 1:49 PM, Gianluca Cioci <gianluca.ci...@gmail.com> > wrote: > >> Dear All >> >> The ion exchange has the great advantage ,over other tecniques, to >> concentrate the protein. >> >> Histrap+sec is a big classic in protein purification but times it is >> worth considering other schemes. Why doing a SEC? It is for removing >> aggregates or a contaminant ? It the latter case probably an ion exchange >> or an Hic would do a better job. For fast changing of the buffer desalting >> with a big column is the way to go. >> Good luck >> Gia >> >> Il 17 Nov 2017 18:57, "David Blum" <dlb...@gmail.com> ha scritto: >> >>> Hi Liuqing, >>> >>> I would not recommend SEC. SEC does not give that great of a separation >>> unless your contaminant is greatly different in size. Instead of SEC, you >>> might want to consider hydrophobic interactions chromatography (HIC). You >>> can add your ammonium sulfate directly to your eluted protein from your >>> IMAC column or IEX, avoiding the dialysis step. I would also recommend >>> trying some test kits which have a variety of columns to test and see what >>> works best for your protein. We use these kits routinely for our clients >>> and have good luck with HIC. >>> >>> >>> >>> *David L. Blum, Ph.D.* >>> Department of Biochemistry and Molecular Biology | *Director, >>> Bioexpression & Fermentation Facility* >>> >>> 120 Green Street | Life Sciences Bldg A414A | Athens, GA 30602 >>> 706-542-1035 <7065421035> | b...@uga.edu | bff.uga.edu >>> >>> On Fri, Nov 17, 2017 at 8:44 AM, Liuqing Chen <519198...@163.com> wrote: >>> >>>> Hello everyone! >>>> I have listened someone suggested that, first use affinity >>>> chromatography (Ni-NTA), then use SEC (superdex200 increase), and finally >>>> used ion exchange (monoQ), to purified protein, which will be used to >>>> crystallization. >>>> My question is why the monoQ used in the finally step, why not the >>>> SEC used at the finally step? >>>> >>>> sincerely >>>> Liuqing Chen >>>> >>> >>> > -- Dr. Nishant Kumar Varshney, Research Associate, C/O Dr. Sameena Khan, Drug Discovery Research Center, Translational Health Science and Technology Institute (THSTI) NCR Biotech Science Cluster, 3rd Milestone, Faridabad – Gurgaon Expressway, Faridabad – 121001 (HARYANA), India Ph: +91- 0129-2876477 Mob: 8390564690
