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Protease & Phosphatase Inhibitors


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#1 Mighty Mouse

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Posted 17 November 2009 - 08:25 AM

I was just wondering what various people used for protease/phosphatase inhibitors in their ChIP during sample prep. I've been using the Millipore kit which comes with a protease inhibitor cocktail, albiet not in a large enough amount, so I've been searching for something else to use. I know Sigma makes several protease/phosphatase inhibitors, however I've come across a product from Pierce that is a combination protease/phosphatase inhibitor (http://www.piercenet...FC-572566DA6E04). Just wondering if anyone has successfully used this, it seems like a pretty easy way to kill lots of birds with one shot.

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MM
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#2 KPDE

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Posted 17 November 2009 - 09:48 PM

I was just wondering what various people used for protease/phosphatase inhibitors in their ChIP during sample prep. I've been using the Millipore kit which comes with a protease inhibitor cocktail, albiet not in a large enough amount, so I've been searching for something else to use. I know Sigma makes several protease/phosphatase inhibitors, however I've come across a product from Pierce that is a combination protease/phosphatase inhibitor (http://www.piercenet...FC-572566DA6E04). Just wondering if anyone has successfully used this, it seems like a pretty easy way to kill lots of birds with one shot.

Thanks,

MM


I don't know about that particular cocktail but in my experience ChIP is pretty robust with respect to the inhibitors. One potential issue with a cocktail that includes phosphatase inhibitors is that the peptides used for generating non-phospho-specific antibodies are not phosphorylated. This means that preserving phosphorylations could potentially block some epitopes. I haven't had this experience as most antibodies that I have tried that don't work, won't work regardless of the presence of phosphatase inhibitors, but it is at least theoretically a possible problem.




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