Crystallizing protein sitting in PBS

From: Jayakrishnan Nandakumar
Date: 15 November 2011 23:25


Hi All,
I have an RNA-binding protein that I can purify out of bacteria in PBS (Phosphate buffered saline;137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4), but which is insoluble in Tris/NaCl-based buffers. My guess would be that the inorganic phosphates (by mimicking RNA) are binding the protein to keep it in solution. My question is whether I can leave the protein in a phosphate-based buffer (at lower salt maybe) to set up crystallization trials or are PBS-based buffers not suitable for crystallization in general. I have always used Tris/NaCl based buffers in the past.

Thanks in advance for your suggestions.
Regards,
JK

----------
From: <Herman.Schreuder

Dear JK,

 

In principle, there is nothing against screening in phosphate buffer. The only reason it is not popular with crystallographers is that phosphate likes to form all kinds of salt crystals, e.g. with calcium. So if you screen, you should be prepared to see salt crystals as well. However, if your protein is happy in phosphate and unhappy in Tris buffer, I would do it, but keep the phosphate concentration as low as possible. 12 mM is already pretty low.

 

Good luck!

Herman

----------
From: Tom Murray-Rust


Hi JK,

As mentioned, phosphate salts are the main disadvantage - you can get
round this by setting up two drops per well: one with your protein in
PBS, and the other with PBS only. That way you should be able to
quickly identify any hits that are due to salt, and which are likely
to be your protein.

Tom

http://twitpic.com/photos/tmurrayrust


----------
From: Shilong Fan


Of course PBS should not be a first choice for screning crystal.

But I will try all kinds of buffer until I got the structure.

Nobady can tell you that you can't get crystal in the PBS buffer. and I will shot everything I have if I have enough beam time.

Good luck.

----------
From: Gloria Borgstahl


A thing we frequently forget is that phosphate can be a precipitating agent
try a phosphate grid screen, just like you would with ammonium sulfate.
If your protein likes PBS, it may want to crystallize with phosphate

See Enrico Stura's footprint screen for example

----------
From: Ed Pozharski


Just a footnote to Tom's excellent comment:

It is possible to have actual protein crystals to grow alongside salt
crystals.  It is also possible for salt crystals to grow only when
protein is present and not with protein storage buffer.  I've seen both
things happening.  My point is that seeing salt crystals in the "buffer
drop" is suggestive, but not an absolute proof.  It stinks to fish out a
(usually heavy and unyielding) salt crystals just to see three
super-bright spots, but that is the only 100% conclusive way I know
(channel-penetrating dyes and UV microscopy have their limits).

On a more informative note, I used protein in PBS once (not because it
was unstable, but rather because I was lazy), and it surprised me how
few salt crystals I got with standard screens.  I tend to think
phosphate will be trouble with divalent cations (calcium and magnesium
primarily), but I presume the solubility is somewhat pH dependent and
that alleviates the problem to some extent.

And of course, in the described scenario of protein that needs phosphate
for solubility there isn't much choice. (But perhaps buffer screening is
in order).

Cheers,

Ed.

--
Hurry up, before we all come back to our senses!
                         Julian, King of Lemurs