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

Vinod Agarkar via proteins%40net.bio.net (by agarkarvi from yahoo.com)
Thu Feb 21 19:32:30 EST 2008



    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 <brighttomorrow from gmail.com> 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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