1 reply to this topic

[Mighty Mouse]

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

[KPDE]

Mighty Mouse, on Nov 17 2009, 08:25 AM, said:

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.