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Not getting ATM acetylation after IR


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#1 JDSBlueDevl

JDSBlueDevl

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Posted 11 March 2015 - 07:26 PM

I'm getting efficient pulldown of ATM from 300 ug of protein with 4 ug anti-ATM antibody.  However, when I try to probe for acetylation after 6 Gy IR (1 hr post-incubation), I do not see any acetylation, even though it has been reported in the literature repeatedly.  I am using an Upstate anti-acetyllysine antibody that is also used for ELISA after HAT assays.  I am using different sources of protease and phosphatase inhibitors than were mentioned in the literature (I am using cOmplete and PhosStop from Roche, with no use of DTT or PMSF), although I fail to see how they would make a difference.  I had read that in ChIP, people use sodium butyrate to inhibit deacetylases, but I am not sure whether these deacetylases would also affect acetylations on non-histone proteins (especially since I have not seen this as a component in lysis buffers outside ChIP).  Should I be using sodium butyrate in my preparations?  Also, when I do a HAT assay, should I make sure to add sodium butyrate to my assay buffer as well?






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