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Proper HAT assay procedure

HAT immunoprecipitation TIP60 acetyltransferase

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#1 Hic5Prostate

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Posted 04 December 2014 - 06:33 PM

I'm using the Abcam colorimetric HAT assay kit work my immunoprecipitations because the Upstate/EMD ELISA-based kit isn't being manufactured anymore. The cell line I'm using, 184A1, grows very slowly, so I can only use 2.5M cells per experiment, which generate 275-300 ug protein per IP for two IPs (ATM and IgG). I performed the HAT assay incubation directly on the beads, as elution with glycine-based buffer was impossible. After a 3-hr incubation on a thermomixer at 37 C, I transferred the reaction mixtures to a 96-well plate for reading. However, what I noticed was that the readings for the IgG IPs correlated nearly exactly with the ATM IPs, indicating that the increase in HAT activity due to Tip60 bound to ATM that I should be seeing (which would agree with previous data from the Price lab at Dana-Farber) is apparently an artifact. Does anyone know whether incubation of HAT reaction mixture with agarose beads containing Protein A/G can interfere with a colorimetric assay? Also, how can I boost growth of 184A1 cells to generate high amounts of protein if it is believed that high protein input can overcome the noise? 1.2*10^8 cells per full experiment seems like a high barrier to my experiment for a slowly growing line.





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