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