[Protein-analysis] Zinc Finger Question

Kristina Blake kab69 at cornell.edu
Tue May 23 20:06:17 EST 2006


Hi-
  I am trying the purify a microtubule binding protein that also 
contains a putative zinc finger.  I had asked some questions before 
about this protein as I am having trouble with it crashing out of 
solution and gotten some good suggestions.
 When I dialyze it into 80 mM PIPES, 1 mM EGTA, and 1 mM MgCl2 pH 6.8 it 
crashes out of solution (changing to PIPES from a 20 mM Tris solution, 
no salt) Anyways, I stil have not answered the question of why it 
crashes out of this solution, I think it may be the Mg but experiments 
changing one condition at a time (ie no MG, or no EGTA, etc, etc.) have 
not been entirely able to answer the question. The protein appears to 
not even be entirely soluble in PIPES by itself. Also changing the pH up 
to 7.5 didnt' seem to help, nor did adding NaCl up to 150 mM (I didn't 
go higher).  I am wondering if anyone knows wether proteins containing 
zinc are even soluble in PIPES.

Also:
      I am wondering if anyone has used TEV protease to cleave the His 
tag off a protein containing zinc.  The normal TEV cleavage buffer is 50 
mM Tris, 0.5 mM EDTA and 1 mM DTT pH 8.0 which are "not friendly" to 
zinc containing proteins.  So I tried a "zinc finger friendly" buffer 
for cleavage and found my protein crashed out of solution in this buffer 
(note: it doesn't crash out in the "normal" TEV cleavage buffers, 
although I have been warned that the EDTA and DTT present might be bad 
for the protein). The zinc finger friendly buffer was as follows: 20 mM 
Tris-HCl pH 7.4, 10µM ZnCl2, 200mM NaCl, 5 mM citrate, 5 mM BME.
    Does any one have any thoughts or suggestions on this?  I'm not sure 
what about this "zinc finger friendly"buffer caused my protein to crash 
out.  Anyone notice anything common between the PIPES, EGTA, Mg buffer 
and this one since my protein crashes out in both. They both have metals 
in the form of Zn and Mg. 
     I really need my protein to be soluble in the above mentioned PIPES 
buffer since I need to be in this buffer to perform microtubule assays 
with my protein of interest. Also a HEPES buffer and MES buffer could 
work, as microtubule assays have also been performed in those buffers. 
Anyone know if zinc fingers are soluable in HEPES or MES? 
  Thanks again for any suggestions, I really dont' know much about 
protein purification and I am super bummed that my protein is entirely 
purifyable and in good concentration right up to the point where I try 
to dialyze it into my working buffer!
  Thanks,
       Kristy Blake



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