Phosphatase Inhibitors into IP lysis buffer! - (Aug/13/2006 )

Hello!

I have been doing some IP work..
And now I want to add phosphatase inhibitor into my lysis buffer..

Im going to use sodium orthovanadate at 0.1mM and beta-glycerophosphate at 40mM..

I have two products from Sigma..

For the sodium orthovanadate, Im going to make 200mM stock in water and

add to lysis buffer fresh each time.. (1:2000 dilution)..

The informations sheet says to heat the solution (in water) at pH 10...

Is this really necessary? And can I store the stock solution on the bench?

Beta-glycerophosphate, I am really lost...

It has a molecular formula of (HOCH2)2CHOP(O)(ONa)2 . xH2O

And the molecular weight is 216.04 (anhydrous basis)...

What is this mean? anhydrous basis....

I need to use the drug at 40mM in lysis buffer.. and I guess I need to add

it fresh each time...

Can anybody please please help me how I should make stock solution?
(with details.. grams... volumes.. since im really lost... )

Thank you Thank you so much in advance.

I usually make stock solutions of 100x or 1000x.

If u need 40mM in ur buffer, make 4M solution and u could use it as 100x.

eg. 1.7283 grams in 2ml, gives u 4M sol.

Use 10ul of the stock for 1ml of the lysis buffer.

Thank you so much!!

It has been great help! =)

I was just worried because in the information sheet, it says the

solubility is 50mg/ml in water...

If I make 4M stock, should I aliquot and freeze at -20oC?

And sodium vanadate also..

Do you know if I need to dissolve it at pH 10? or just normal water (like milliQ)?

And should I store at RT or freeze it?

Thank you so much for your help!!