Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
- - - - -

Not getting ATM acetylation after IR

  • Please log in to reply
No replies to this topic

#1 JDSBlueDevl



  • Active Members
  • PipPipPipPipPip
  • 39 posts

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?

Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.