Sodium Orthovanadate kinetics in media incubations - (Oct/11/2007 )
Our current protocol involves a one minute, five minute, ten minute, and one hour incubation of cells with treatment and control media both supplemented with sodium orthovanadate. We are repeatedly seeing a strong phosphorlyation signal in our one minute sample and one hour sample. However the five minute time point is incredibly faint and the ten minute is non-existent.
How is this possible if sodium orthovanadate is preventing phosphatase activity?
Does the activity of sodium orthovanadate wear off after a few minutes at 37C?
Any help from anyone with knowledge about sodium orthovanadate would be great.
How is this possible if sodium orthovanadate is preventing phosphatase activity?
Does the activity of sodium orthovanadate wear off after a few minutes at 37C?
Any help from anyone with knowledge about sodium orthovanadate would be great.
what is the reason to use ortho-vanadate for cells? is it to block phosphate transporters?
I should have been more specific. We are lysing the cells and measuring phosphorylation of an ITAM, our lysis buffer also contains sodium orthovanadate.
so you use it as phosphatase inhibitors; I think, but may be wrong, that ortho-vanadate is more specific to phospho-Y phosphatases; it appears to be sterically similar to phosphoryls and blocks the catalytic site of phosphatases;
commonly used in lysis buffers to save phosphorylations...