How to keep protein in Phosphatased Form - Run Co-IP need to have protein in phosphatased form (Nov/13/2007 )
I need to run a Co-IP which can tell me, does my protein work with certain substrate at it phosphated or non-phosphated form? Is there any chemical I can use to ensure that? I know I can use NaF to keep them in non-phosphataed form ... But what about the Phosphated one?
-vio-
QUOTE (vio @ Nov 13 2007, 03:17 AM)
I need to run a Co-IP which can tell me, does my protein work with certain substrate at it phosphated or non-phosphated form? Is there any chemical I can use to ensure that? I know I can use NaF to keep them in non-phosphataed form ... But what about the Phosphated one?
NaF is an old-fashioned broad inhibitor of phosphoprotein phosphatases; if you like to save phosphorylation, add NaF (if you have no better PPP-inhibitor)
-The Bearer-
QUOTE (The Bearer @ Nov 14 2007, 04:22 AM)
QUOTE (vio @ Nov 13 2007, 03:17 AM)
I need to run a Co-IP which can tell me, does my protein work with certain substrate at it phosphated or non-phosphated form? Is there any chemical I can use to ensure that? I know I can use NaF to keep them in non-phosphataed form ... But what about the Phosphated one?
NaF is an old-fashioned broad inhibitor of phosphoprotein phosphatases; if you like to save phosphorylation, add NaF (if you have no better PPP-inhibitor)
It would be better to run along side with an assay to make sure no phosphorylation is saved ... right? What do I need to add?
-vio-
QUOTE (vio @ Nov 13 2007, 07:15 PM)
QUOTE (The Bearer @ Nov 14 2007, 04:22 AM)
QUOTE (vio @ Nov 13 2007, 03:17 AM)
I need to run a Co-IP which can tell me, does my protein work with certain substrate at it phosphated or non-phosphated form? Is there any chemical I can use to ensure that? I know I can use NaF to keep them in non-phosphataed form ... But what about the Phosphated one?
NaF is an old-fashioned broad inhibitor of phosphoprotein phosphatases; if you like to save phosphorylation, add NaF (if you have no better PPP-inhibitor)
It would be better to run along side with an assay to make sure no phosphorylation is saved ... right? What do I need to add?
may be I miss the point: Do you like to save phosphorylations or try to get rid off it?
-The Bearer-
QUOTE (vio @ Nov 13 2007, 09:15 PM)
QUOTE (The Bearer @ Nov 14 2007, 04:22 AM)
QUOTE (vio @ Nov 13 2007, 03:17 AM)
I need to run a Co-IP which can tell me, does my protein work with certain substrate at it phosphated or non-phosphated form? Is there any chemical I can use to ensure that? I know I can use NaF to keep them in non-phosphataed form ... But what about the Phosphated one?
NaF is an old-fashioned broad inhibitor of phosphoprotein phosphatases; if you like to save phosphorylation, add NaF (if you have no better PPP-inhibitor)
It would be better to run along side with an assay to make sure no phosphorylation is saved ... right? What do I need to add?
phosphatase.
-mdfenko-