Preparation of sodium orthovanadate solutions

[admin]

Hi All
I'm in dire need of some advice on the preparation of a sodium orthovanadate solution used as a cell culture treatment to inhibit protein phosphotyrosine phosphatases. I've heard of two methods: making it up in water, pH10, boiling and then readjusting to pH10 to ensure presence of monomers as well as making it up fresh just before addition to cells. If anyone has any advice on the best method to use, I'd be very grateful.
Ciao

posted by micheline (please re-register thanks!)

[danfive]

I use sodium orthovanadate from Sigma (#S6508) and prepare it just as you described. Making a stock bottle of ultrapure water at pH10 is the most useful step. Boiling works very fast and if the chemical is fresh it may change its initial yellow color to clear once added to solution, just boil it for a few minutes to be sure all the decavanadate is converted. I freeze mine in epi-tubes and throw out any remaining solution after the first thawing, but preparing it fresh is probably the better technique.

[mickey07]

Thanks for your reply, Danfive. I have decided to make the vanadate up fresh everytime and boiling it for a few minutes before use. Thanks for the very useful tip!!!!
Cheers

[danfive]

Here is a link to an article showing the benefit of using NaV to inhibit lysosomal degradation.
[Only registered users see links. ]

[vinnnervinayak]

Hi Mickey.
I have tried ths protocol to make activated sodium orthovanadate. However when i do add HCL to the 100-200mM solution to adjust ph to 10 it does turn yellow.However i tried boiling it for several minutes (20-30) and it still did not turn colourless.any suggestion in this regard would be really helpful !

vinayak

[danfive]

Originally Posted by vinnnervinayak

Hi Mickey.
I have tried ths protocol to make activated sodium orthovanadate. However when i do add HCL to the 100-200mM solution to adjust ph to 10 it does turn yellow.However i tried boiling it for several minutes (20-30) and it still did not turn colourless.any suggestion in this regard would be really helpful !

vinayak

How about making Water at pH 10.
Then using the pH 10 water to dissolve the orthovanadate.
Once dissolved insert the bottle or tube into boiling water bath for 10 min or until the yellow color disappears (tends to happen immediately with fresh ortho).

Just so you know yellow solution is actually decavanadate, the wrong configuration to inhibit lysosomal enzymes. So you need to get it clear (orthovanadate configuration) by boiling it.

If you cannot get the ortho to be clear by changing the steps for preparation, then you need to buy fresher orthovanadate.

[wr_1a]

thanks

[Crunchy Nut]

Originally Posted by vinnnervinayak

Hi Mickey.
I have tried ths protocol to make activated sodium orthovanadate. However when i do add HCL to the 100-200mM solution to adjust ph to 10 it does turn yellow.However i tried boiling it for several minutes (20-30) and it still did not turn colourless.any suggestion in this regard would be really helpful !

vinayak

I found that sometimes the solution doesn't go colourless after the first boil. Ignoring this and readjusting the pH to 10 anyway, before boiling again, seemed to do the trick.