Making Phosphate buffer with calcium chloride - (Apr/13/2012 )

I am trying to make phosphate buffer with calcium but I always see a significant white precipitate in the buffer. Is there a way to avoid this precipitates? How do people make PBS, PB, etc with calcium without precipitation? My buffer is:

50mM Sodium Phosphate, pH 7.4,
0.5 M NaCl and
0.5mM CaCl2

Trying to purify a calcium dependent protein and calcium should be present. Also phosphate is better for the purification procedure.

Your responses and suggestions are very much welcome

Hi,
Found this in the archive about the same question posted by someone else (http://www.protocol-online.org/biology-forums/posts/39878.html).

Unfortunately, there was no reply regarding the outcome the suggested preparation. Good luck.

you will have to eliminate the use of phosphate buffer in your purification scheme (unless you use hydroxyapatite as a purification step).

you can't use calcium with phosphate buffers and expect any significant amount of free calcium.

Maybe it's better to use tris-HCl

Instead of using CaCl2 (FW 110.98), try CaCl2+2H2O (FW 147.01), you'll see the difference.

newbie99 on Wed Apr 25 18:14:56 2012 said:

hydration of the calcium chloride makes no difference, calcium phosphate will still precipitate.

The order is important. You add in three ingredients all at once, you'll get precipitation.

you'll get precipitation regardless.

Thank you all for your replies. It is not clear how to avoid the precipitates. Does it mean PBS with Mg2+ and Ca2+ is useless interms of availability of Ca2+?

I will try other buffer systems.

Thank you all for giving your opinioins

I make PBS+/+ all the time and I never see precipitates.