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-   -   [Protein-analysis] Recombinant Protein precipitate out under 20mM KPB/100 mM NaCl ??? (http://www.molecularstation.com/forum/protein-forum/27019-%5Bprotein-analysis%5D-recombinant-protein-precipitate-out-under-20mm-kpb-100-mm-nacl.html)

Vinod Agarkar 02-22-2008 12:32 AM

[Protein-analysis] Recombinant Protein precipitate out under 20mM KPB/100 mM NaCl ???
 


Hi,

May be you could try followings:

1) While collecting fractions from Ni-NTA column, keep 0.25 to 0.5mM EDTA in collection tubes.
2) Dialyse against the buffer (elution buffer) for 2-3 hrs.
3) Now, make 20mM KPB and add slowly with intervals 20-30min to decrease the NaCl concentration (I will prefer to do in cold). You will know where your protein is happy, I mean what salt concentrations.
4) Changing buffer like Tris or anything is not a bad idea.
5) Check with KCl instead of NaCl or combinations of two.Or may be you need some other additives.

Have a nice time.
Vinod Agarkar

Nico <[Only registered and activated users can see links. Click Here To Register...]> wrote:
Hi,

I am changing buffer for my protein, which was purified from Ni-NTA column.
Under 20mM KPB/500mM NaCl, it was good. But when change to 20 mM KPB/100 mM NaCl,
the protein precipitate out. What should I do? Change to Tris-HCl Buffer or.....

Please help me out,

Many thanks,

Nico


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