Thread: Sodium deoxycholate solubility issues - HELP!

Hi there,

I really hope someone out there can help me on this... We currently have quite an annoying problem in the lab. We try to use sodium deoxycholate as solubilizer for membrane proteins in a buffer intended to use for hydrophobic interaction chromatography in protein purification.

The final deoxycholate concentration is supposed to be 0.5%. See complete composition below:

10 mM NaH2PO4, 1 mM EDTA (Na2*2 H2O), 0.5 M NaCl, 20 % Glycerol, 0,5% Na Deoxycholate; pH 7,4

This buffer has routinely been used without any solubility problems in our lab before and we are all now extremely surprised to see that we do not get the deoxycholate into solution anymore. Three different people have tried to prepare the buffer - without success. We have tried several things:
1. We adjusted pH to 7.4 before adding the deoxycholate (which is supposed to precipitate at pH lower than 6.9 or so) - it still precipitates.
2. We adjusted everything to pH 10 before adding the deoxycholate whcih dissolved at that pH and, after addition, titrated the pH down to pH 7.4 - the deoxycholate precipitated.
3. We used ultrasound or heat to dissolve it - this works at first but then the solution becomes gel-like (not very suitable if you want to use it as the mobile phase in chromatography). :?
4. We dissolve everything first and then combine the solutions - deoxycholate still precipitates. Funnily, deoxycholate is perfectly soluble up to 10% in plain water.

However, when we used an old stock solution of deoxycholate which had been sitting in the cold room for about a year and which most probably derives from another supplier (of course nobody here has a clue which one we used to use) - everything is perfectly dissolved. That stock solution is gone now, though, and we really need to find out what's going wrong here.

Please help me, I appreciate any thought on this!

Best regards from Kiel, Germany
Claudia

Hi,

The deoxycholate is forming micelles that gets worst with high salt concentration. The critical micelle concentration is 2 mM. 0.5% is about 12 mM. I would suggest trying to solubilize without the NaCl.

Arthur

Originally Posted by Claudia266

Hi there,

I really hope someone out there can help me on this... We currently have quite an annoying problem in the lab. We try to use sodium deoxycholate as solubilizer for membrane proteins in a buffer intended to use for hydrophobic interaction chromatography in protein purification.

The final deoxycholate concentration is supposed to be 0.5%. See complete composition below:

10 mM NaH2PO4, 1 mM EDTA (Na2*2 H2O), 0.5 M NaCl, 20 % Glycerol, 0,5% Na Deoxycholate; pH 7,4

This buffer has routinely been used without any solubility problems in our lab before and we are all now extremely surprised to see that we do not get the deoxycholate into solution anymore. Three different people have tried to prepare the buffer - without success. We have tried several things:
1. We adjusted pH to 7.4 before adding the deoxycholate (which is supposed to precipitate at pH lower than 6.9 or so) - it still precipitates.
2. We adjusted everything to pH 10 before adding the deoxycholate whcih dissolved at that pH and, after addition, titrated the pH down to pH 7.4 - the deoxycholate precipitated.
3. We used ultrasound or heat to dissolve it - this works at first but then the solution becomes gel-like (not very suitable if you want to use it as the mobile phase in chromatography). :?
4. We dissolve everything first and then combine the solutions - deoxycholate still precipitates. Funnily, deoxycholate is perfectly soluble up to 10% in plain water.

However, when we used an old stock solution of deoxycholate which had been sitting in the cold room for about a year and which most probably derives from another supplier (of course nobody here has a clue which one we used to use) - everything is perfectly dissolved. That stock solution is gone now, though, and we really need to find out what's going wrong here.

Please help me, I appreciate any thought on this!

Best regards from Kiel, Germany
Claudia