Hi,I'm working on some pull-downs using epitope tagged recombinant proteins mixed with 3t3 whole cell lysate, and I am trying to test nucleotide dependence of the interaction.
I am planning on using apyrase to remove basal levels of ATP/ADP from my whole cell lysate. After apyrase treatment, I will then add my test nucleotide conditions (ie. ATP, ATPgS and so forth). I just received apyrase from NEB, but try as i might, I cannot find a good protocol for this. I figure that after the apyrase reaction is completed, it must be quenched somehow, otherwise my added nucleotides will be degraded.
Does anyone have experience with this or can you suggest a protocol for me?
Thanks a lot!
Apyrase treatment of whole cell lysate
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