phosphatase inhibitor cocktails... - do you use them or a preffered one? (Oct/25/2006 )
Hi.
I'm quite tough with phophatase inhibition and therefore need help to make a good choice.
I need to analyze a Tyrosine phosphate on STAT1 by western blot. Basically, sodium orhivanadate is used for phophatases blocking in extraction buffers.
But it seems cocktails offer more confidence for that.
So i'be done some surfing in companies sites, but would have your advices too. Do you have bad experieces or difficulties wth a cocktail? or in contrary your favorite?
Thanks for any comments.
never tried cocktails,
I used orthovanadate, and was satisfied. I worked on ERK JNK p38 and FAK phosphorylation.
I wouldn't bother buying one.
In addition to OV, you can add NaF and even Beta-glyercophosphate. I would think these are sufficient.
[quote name='fred_33' date='Oct 25 2006, 07:01 PM' post='74476']
Hi.
I'm quite tough with phophatase inhibition and therefore need help to make a good choice.
I need to analyze a Tyrosine phosphate on STAT1 by western blot. Basically, sodium orhivanadate is used for phophatases blocking in extraction buffers.
But it seems cocktails offer more confidence for that.
So i'be done some surfing in companies sites, but would have your advices too. Do you have bad experieces or difficulties wth a cocktail? or in contrary your favorite?
Thanks for any comments.
[
a broad unspecific Y-phosphatase inhibitor such as o-vanadate (which has site effects on some ATPases and other phosphatases) may indirectly inhibit or force other reactions to phosphorylate/dephosphorylate your STAT1;
I see two possibilities for optimization:
extraction without YPPP-inhibitor, and simulation of phosphorylation/dephosphorylation kinetcis of STAT1 in vitro by testing various specific YPPP-inhibitors independently (to get a clue which YPPP or family of YPPP is responsible)
or if you know the specific YPPP for the YP-site of STAT1, try to get a specific inhibitor for this YPPP and use it in your extraction buffer; might be an expensive story, so try micro-extractions
hi,
try one of these "specialized" phosphatase inhibitors cocktails from Calbiochem
http://www.merckbiosciences.co.uk/html/cbc...r_Cocktails.htm
my blots are cleaner since i use set II and it is not more expensive than Na3VO4 powder!
Sebastien