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OKADAIC ACID for protein prep


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#1 yoshimi79

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Posted 08 April 2010 - 08:47 AM

Has anyone used okadaic acid as a phosphatase inhibitor when prepping protein from cell or tissue homogenates (for use in westerns)? I have a few questions

1. What concentration is necessary for PP1/2 inhibition? I've seen it used at multiple concentrations...

2. Is it possible to activate some kinase activity (it's been published that okadaic acid can activate PKC activity) in homogenized tissue that could confound the data. I keep my samples on ice and add 1mM EDTA, but I'm afraid that if phosphatases are inhibited without proper inhibition of kinases, the phosphorylated protein signal could be inflated.

3. How worried should I be about working with okadaic acid? It has a rather daunting MSDS....Any specific recommendations on handling?

Thanks.
Cheers.

#2 Inmost sun

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Posted 13 April 2010 - 04:31 AM

Has anyone used okadaic acid as a phosphatase inhibitor when prepping protein from cell or tissue homogenates (for use in westerns)? I have a few questions

1. What concentration is necessary for PP1/2 inhibition? I've seen it used at multiple concentrations...

2. Is it possible to activate some kinase activity (it's been published that okadaic acid can activate PKC activity) in homogenized tissue that could confound the data. I keep my samples on ice and add 1mM EDTA, but I'm afraid that if phosphatases are inhibited without proper inhibition of kinases, the phosphorylated protein signal could be inflated.

3. How worried should I be about working with okadaic acid? It has a rather daunting MSDS....Any specific recommendations on handling?

Thanks.
Cheers.


1. take at least the 10-fold of Ki or IC 50 which means at least 100-150 nM;
2. I do not think that OA directly activates PKC but indirectly by blocking PP1/PP2A, and, therefore, increase of PKC phophorylation
3. You work in low dosis however swallowing of OA may cause diarrhea; use gloves




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