serine/threonine phosphatase inhibitors - what are the commonly used ones (Oct/05/2004 )
Hi, i'm working with some phosphorylation related activity, and i would like to inhibit phosphatase activity during the preparation of cell lysate. Any good advice on what inhibitors to use and proper concentration in the lysis buffer? thanks a million!!
1-Naphthyl phosphate sodium salt: is a broad inhibitor of phosphatase.
Na3VO4: typicaly tyrosine phosphatase inhibitor
Okadaic Acid: Ser/Thr phosphatase inhibitor, such as PP1 and PP2
For others, you can search them in Biochem company, where you can find a set of phosphatase inhibitors.
I use Na3VO4 and okadaic acid as well. I also use NaF, and i usually incorporate sodium phosphate in my solubilisation buffer, wich is a false substrate for the phosphatases, and also EDTA, since phosphatases usually need calcium.
I have not an answer, but more questions in this topic.
I start to work with protein phosphorylation and currently using mixture Na3VO4, NaF, okadaic acid and EDTA. But I found a reference (Pendergast AM, Venema RC, Traugh JA., J Biol Chem. 1987 May 5;262(13):5939-42.) that b-glycerophosphate (160 mM) is more potent inhibitor of wide range of the phosphatases. I have purchase from Sigma "Glycerol phosphate disodium salt hydrate", G6501, but it is even not toxic at all according MSDS! How it can be?! Does anybody use b-glycerophosphate as a phosphatase inhibitor? And from which company you purchase it?
Many thanks in advance!
Hi, phosphome and Oleksii.
During a couple of years I used a lysis buffer for membrane phospho-proteins and the buffer had B-glicerophosphate. It works very good:
Tris-HCl 10mM pH 7.4, NaCl 50mM, EDTA 5mM, Triton X-100 1%, SDS 0.05%, NaF 50mM, Na3VO4 100microM, B-glicerophosphate 10mM, sodium pyrophosphate 10mM, phospho-serine 1mM, phospho-threonin 1mM, phospho-tyrosine 1mM, leupeptin 20microg/ml, bacitracine 500microg/ml, trypsin soybean inhibitor 50microg/ml and PMFS 100microg/ml.
All reagent were from Sigma and must be prepared fresh before use it.
I would advice you to look for promega's phosphatase inhibitor kit. They have specific inhibitors for serine/threonine or tyrosine phosphatases.