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Phosphatase cleaving problems
Posted by: carnegies (IP Hidden, New member, 1)
Date: August 4, 2005 10:59AM
Hello all,
I am doing an experiment where I am labeling my protein with P-32 and S-35 (successful) and subsequently immunoprecipitated from host cells (successful). My problem is when I add calf intestinal alkaline phosphatase (CIAP) to the bead-bound complex and then do an SDS-PAGE > autoradiography > phosphorimaging .. our protein shows up fine which just shows that phosphorus remains bonded to our protein. My question is this: why is the phosphatase not working and under what conditions will the phosphatase not cleave off phosphorus from a protein. I am stumped!
Re: Phosphatase cleaving problems
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 4, 2005 01:36PM
It sounds as if your phosphatase is being inhibited or has lost activity. the type of phosphatase affects what inhibits it. In general phosphatases are inhibited by sodium orthovanadate and sodium stibogluconate (used in PBS buffers). any source of phosphorus in your experiment can also affect the desired phosphatase reaction, as it provide additional substrate for the enzyme to degrade thus effectively diluting the phosphatase. you may wish to add additional phosphatase to the bead-bound complex. i would also suggest doing a lit search for your particular phosphatase to see if it has additional inhibitors.
Re: Phosphatase cleaving problems
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: August 4, 2005 02:01PM
Do you know your proteins are Tyr-P and/or The/Ser-P??
CIAP may not work for both. You need to pick a tyrosine phosphatase and/or a threonine/serine phosphatase or combine both.
Re: Phosphatase cleaving problems
Posted by: mito lab (IP Hidden, Unregistered user, )
Date: August 4, 2005 03:08PM
I am having the same question. Is the effect of CIAP specific or it dephosphorylate any substrate including DNA and p-Tyr, p-Threonine/Ser? Also, can shrimp alkaline phosphotase (SAP) be used to dephosphorylate p-Threonine/Ser same as CIAP? Is the buffer for dephosphorylation of DNA necessary for protein treatment? Thank you.
Re: Phosphatase cleaving problems
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 4, 2005 03:33PM
CIAP is not specific as it dephosphorylates serine, threonine and tyrosine residues in proteins. what pH is your solution? it needs to be alkaline for CIAP to work properly. is it possible you CIAP is old? remember CIAP is only stable for one year and must be stored at -20C.
Re: Phosphatase cleaving problems
Posted by: mito lab (IP Hidden, Unregistered user, )
Date: August 4, 2005 04:51PM
So it is necessary to use the CIP buffer included with the enzyme to make the pH optimal for CIAP (I have not checked the buffer recipe). Thank you for your reply.
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