10 replies to this topic

[kwesi]

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

[lsek]

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

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

[mdfenko]

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.

[Sotirios]

Maybe it's better to use tris-HCl

[newbie99]

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

[mdfenko]

newbie99, on 25 April 2012 - 10:14 AM, said:

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

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

[newbie99]

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

[mdfenko]

you'll get precipitation regardless.

[kwesi]

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

[newbie99]

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

[mdfenko]

newbie99, on 07 May 2012 - 05:55 PM, said:

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

then your concentrations of phosphate and/or calcium are low so the precipitate is not easily detectable and/or you haven't let it sit long enough to see the precipitate.