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: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of Jayakrishnan Nandakumar
Sent: Wednesday, November 16, 2011 12:26 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystallizing protein sitting in PBS
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
