I am trying to detect protein phosphorylation by IP-IB. To prepare tissue lysates I am using RIPA lysis buffer which contains EDTA . I add Sodium Orthovanadate as a phosphatase inhibitor. I see in the data sheet that "The inhibition by Vanadate is completely reversible upon the addition of EDTA".
Does it mean that sodium orthovanadate is inactive when I add it to my RIPA buffer?
Thanks in advance.
It seems that EDTA can bind vanadate*, so it would be pointless to add vanadate as an inhibitor in the presence of EDTA, unless the vanadate concentration is in excess of the EDTA concentration. EDTA itself is a serine/threonine phosphatase inhibitor due to binding Mg2+, which is required for enzyme activity, so vanadate is probably unnecessary anyway.
*http://www.plantphysiol.org/content/75/3/586.full.pdf
''EGTA, on the other hand, forms a weaker complex with vanadate (34) and may therefore be a practical alternative when divalent cations must be chelated''
http://www.jbc.org/content/272/2/843.full
I do agree with Dr. ADam, its of no use adding vanadate when EDTA present in the buffer, my suggestion is that try ur experiment without adding vanadate.
all the best
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