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
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
> 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" 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
