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
